Compositions for improving cells and organisms

ABSTRACT

The present invention provides compositions comprising randomized in-frame fusion polynucleotides and methods for introducing them into a host organism to obtain desirable phenotypic changes that modulate tolerance to stress, thus creating novel characteristics of the transformed organism.

This application is a Divisional application of co-pending applicationSer. No. 15/500,803 filed Jan. 31, 2017, and for which priority isclaimed under 35 U.S.C. § 120. application Ser. No. 15/500,803 is theNational Phase of PCT International Application No. PCT/US15/43285 filedon Jul. 31, 2015 under 35 U.S.C. § 371, which claims priority under 35U.S.C. § 119(e) on U.S. Provisional Application No. 62/031,624 filed onJul. 31, 2014, the entire contents of each of which are herebyincorporated by reference.

FIELD OF THE INVENTION

This invention relates to non-naturally occurring fusion polynucleotidesencoding fusion polypeptides and methods of introducing the fusionpolynucleotides into host organisms to generate new and/or improvedphenotypes.

BACKGROUND OF THE INVENTION

Currently, there is a shift to biological systems for production of avariety of chemicals and fuels, and a wide assortment of organisms areand will be used, most of them microbes, with an increasing tendencytowards photosynthetic organisms (Dismukes 2008). The ability to growrobustly, the ability to resist toxic compounds, abiotic stresses,changes in growth conditions, and efficient production of the materialsand compounds of interest, are desirable properties of these organisms.

Yeasts are widely employed as fermentation organisms for the productionof ethanol, butanol, isobutanol and other alcohols, and a variety ofcommodity and fine chemicals. Yeasts used for bio-production include thebaker's yeast Saccharomyces cerevisiae; other Saccharomyces species;Schizosaccharomyces pombe; Kluyveromyces species such as K. lactis, K.marxianus and K thermotolerans; Candida species such as C. albicans, C.glabrata, C. stellate, C. tropicalis, C. dubliniensis and C. keroseneae;Pichia species such as P. angusta, P. anomala, P. membranifaciens and P.pastoris; oleaginous yeasts such as Yarrowia hpolytica; and other yeastspecies such as Dekkera/Brettanomyces species, Brettanomycesbruxellensis, Torulaspora delbrueckii and Zygosaccharomyces bailii.

Many biological production systems using S. cerevisiae and othereukaryotic and prokaryotic production hosts depend on resistance andtolerance properties of the production organisms for efficientproduction of the desired chemical. For example, many fermentationprocesses are exothermic and require the removal of heat or cooling ofbioreactors to ensure the continued viability and productivity of theorganism used for fermentation. Media used for production can have lowor high pH values (i.e. pH<5.0 for low pH values, pH>9.0 for high pHvalues) due to acidic or basic pre-treatment processes that were usedfor the production of sugars used in the fermentation. Alternatively,media used in fermentation can contain salts (i.e. sodium chloride)resulting from neutralization with bases or acids of the acids and basesused in the pre-treatment processes. Because the growth of many speciesof microbes, including many yeasts, is inhibited by heat, salt, low pHor high pH, it is often necessary to employ microbes that are naturallytolerant or resistant to these abiotic stresses, or to engineersensitive strains and species of microbes for higher levels of toleranceor resistance.

Alcohols, such as ethanol, butanol and isobutanol are common products offermentation processes employing yeasts and other microbes. Alcohols aretoxic compounds and can be tolerated by yeasts and other microbes onlyin limited concentrations. Although some ethanol-producing yeast speciesare naturally resistant to ethanol (for example Saccharomycescerevisiae), higher ethanol tolerance is generally desirable inethanol-producing industrial yeast strains to maximize the productivityof fermentation processes. Other alcohols such as butanol are highlytoxic at low concentrations, and yeast species that are naturallyresistant to these alcohols have not been found. It is therefore broadlydesirable to enhance alcohol tolerance in yeast species used for theproduction of alcohols (Cakar 2012, Doğan 2014).

Furthermore, the feedstocks used in a variety of biological productionsystems, particularly those feedstocks derived from degradation of plantproducts, often contain elevated concentrations of salts, acetate,growth-inhibitory carbohydrates, or various toxic organic compoundsderived from plant lignins. The ability of a production organism totolerate the presence of these toxic compounds is a prerequisite formaximal productivities. In addition, the pH found in production systemsmay be outside the pH optimum for the production organism used, and theorganism's ability to grow at pH values outside of its natural optimummay be important in certain production systems.

Renewable biomass, including lignocellulosic material and agriculturalresidues such as corn fiber, corn stover, corn cob, wheat straw, ricestraw, and sugarcane bagasse, are low cost materials for bioethanolproduction. Dilute acid hydrolysis is commonly used in biomassdegradation which hydrolyzes cellulose and hemicellulose fractions toincrease fiber porosity to allow enzymatic saccharification andfermentation of the cellulose fraction. (Saha 2003). However, acidhydrolysis both acidifies the resulting mix of sugars and also generatesinhibitory compounds that interfere with microbial growth and hinderssubsequent fermentation. These compounds include aldehydes (such asfurfural, 5-hydroxymethylfurfural, etc.), ketones, phenols, and organicacids (such as acetic, formic, levulinic acids, etc.). Two of the mostpotent inhibitors are furfural and 5-hydroxymethylfurfrual(5-hydroxymethylfurfrual referred to as “HMF” hereafter). Yeast growthcan be reduced by the combination of furfural and HMF at concentrationsas low as 5 mM (Liu 2004).

These inhibitors can be removed from the hydrolysate before its use infermentation, using physical chemical or enzymatic steps and treatments.However, these additional steps add complexity to the productionprocess, produce waste products, and add significantly to the productioncost. Alternatively, species or strains of microbes or yeasts need to beused for fermentation that are resistant to inhibitors present in sugarsderived from biomass and other sources, resistant to ethanol and otherproducts of the fermentation, and/or tolerant of abiotic stresses suchas high temperature, high salt and low pH that are frequentlyencountered during fermentation processes (Cakar 2012, Doğan 2014). Mostyeast strains, including industrial strains, are susceptible to thegrowth-inhibiting compounds released by dilute acid hydrolysispre-treatment (Martin 2003). Yet few yeast strains tolerant toinhibitors are available and the need for tolerant strains is wellrecognized (Klinke 2004, Zaldivar 2001, Nieves 2015).

Genetic or epigenetic changes in organisms can improve the organisms'performance and raise their productivities. Particularly useful is theintroduction of nucleic acids that confer dominant traits. This impliesthat the functions performed by the introduced nucleic acids, whetherthey encode RNA or protein or perform another function in the cell ororganism, alter or overrides the function performed by similar nucleicacids that are naturally present in the cell or organism.

The present disclosure describes 83 fusion polynucleotides that conferresistance and tolerance in S. cerevisiae to alcohols and to abioticstresses such as heat, salt, and low pH. These fusion polynucleotidesare generated by pairwise fusion of full length open reading framespresent in the S. cerevisiae genome. They are useful for improving S.cerevisiae, other yeasts, and other production microbes and to raise theproductivity of a variety of biological production systems.

BRIEF SUMMARY OF THE INVENTION

The fusion polynucleotides described in the present disclosure wereisolated from fusion polynucleotide libraries prepared by fusing pairsof intact S. cerevisiae open reading frames precisely and in-frame in arandom manner, inserting the pairwise gene fusions into a plasmid vectorcontaining sequences for their propagation in bacteria and in yeast,expressing the fusion polynucleotides in yeast, generating a populationof yeast cells harboring these fusion gene constructs, selecting thepopulation for resistance to a variety of products and stresses,including ethanol, butanol, heat, salt and low pH, and recovering theplasmids from cells surviving the selections as set forth in U.S. Ser.No. 14/134,619. The plasmids were then individually tested for theirability to confer alcohol or stress tolerance in yeast. Of the 83 fusionpolynucleotides described in the present disclosure, 63 were isolatedand tested in this manner (SEQ ID NO: 1-63). Twenty additional fusionpolynucleotides (SEQ ID NO: 205-224) were prepared individually and in atargeted manner by combining open reading frames identified among thefirst 63 fusion polynucleotides with each other, or with additional openreading frames present in the yeast genome, in new sequencecombinations.

Among the fusion polynucleotides listed in the disclosure are multipleones (61 different fusion polynucleotides) containing the yeast WHI2gene (Saul 1985, Mountain 1990, Mountain 1990a, Radcliffe 1997, Kaida2002, Leadsham 2009, Mendl 2011, Müller 2011). WHI2 has previously beenimplicated in stress tolerance (Mendl 2011, Müller 2011) as well as avariety of other cellular processes, including regulation of colonymorphology, control of cell division and cell size, nutritional sensing,and mitochondrial function.

Among the fusion polynucleotides listed in the disclosure are two fusionpolynucleotides and their corresponding fusion proteins which representfusions of the WHI2 protein with phosphatases, 14 fusion polynucleotidesand their corresponding fusion proteins which represent fusions of theWHI2 protein with members of the KEOPS complex, a conserved proteincomplex with a role in telomere maintenance, but of largely unknownfunction (Bianchi 2006, Downey 2006), as well as 11 fusionpolynucleotides and their corresponding fusion proteins which representfusions of the WHI2 protein with members of the EGO complex, a vacuolarmembrane-associated protein complex with a role in activation ofmicroautophagy during exit from rapamycin-induced growth arrest(Dubouloz 2005, Gao 2006, Piper 2006).

The present disclosure describes the sequences of these 83 fusionpolynucleotides, and their activity for conferring stress and alcoholtolerance.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1—Table 5A: Heat and ethanol resistance and tolerance activities of19 fusion polynucleotides and their individual constituent ORFs inglucose-containing medium in S. cerevisiae.

FIG. 2—Table 5B: Butanol and low pH resistance and tolerance activitiesof 19 fusion polynucleotides and their individual constituent ORFs inglucose-containing medium in S. cerevisiae.

FIG. 3—Table 6A: Heat and ethanol resistance and tolerance activities of20 KEOPS/EGO fusion polynucleotides and their individual constituentORFs in glucose-containing medium in S. cerevisiae.

FIG. 4—Table 6B: Butanol and low pH resistance and tolerance activitiesof 20 KEOPS/EGO fusion polynucleotides and their individual constituentORFs in glucose-containing medium in S. cerevisiae.

FIG. 5—Table 7A: Heat, ethanol, butanol, low pH and salt resistance andtolerance activities of 6 fusion polynucleotide GAL1 centromericplasmids and their corresponding individual ORF GAL1 centromericplasmids in galactose-containing medium in S. cerevisiae.

FIG. 6—Table 7B: Heat, ethanol, butanol, low pH and salt resistance andtolerance activities of 9 fusion polynucleotide GAL1 centromericplasmids and their corresponding individual ORF GAL1 centromericplasmids in galactose-containing medium in S. cerevisiae.

DETAILED DESCRIPTION OF THE INVENTION

The following abbreviations and definitions will be used for theinterpretation of the specification and the claims.

As used herein, the terms “comprises,” “comprising,” “includes,”“including,” “has,” “having,” “contains” or “containing,” or any othervariation thereof, are intended to cover a non-exclusive inclusion. Forexample, a composition, a mixture, process, method, article, orapparatus that comprises a list of elements is not necessarily limitedto only those elements but may include other elements not expresslylisted or inherent to such composition, mixture, process, method,article, or apparatus. Further, unless expressly stated to the contrary,“or” refers to an inclusive or and not to an exclusive or. For example,a condition A or B is satisfied by any one of the following: A is true(or present) and B is false (or not present), A is false (or notpresent) and B is true (or present), and both A and B are true (orpresent).

Complementary nucleotide sequence: As used herein, a complementarynucleotide sequence is a sequence in a polynucleotide chain in which allof the bases are able to form base pairs with a sequence of bases inanother polynucleotide chain.

Composite open reading frame: As used herein, a composite open readingframe results from the in-frame fusion of at least two differentstarting open reading frames, resulting in a new open reading framecomprising all starting open reading frames and encoding a fusionprotein comprising the sequences encoded by all starting open readingframes

Control elements: The term ‘control elements’ refers to nucleotidesequences located upstream (5′ non-coding sequences), within, ordownstream (3′ non-coding sequences) of a coding sequence and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences may includepromoters, translation leader sequences, introns, polyadenylationrecognition sequences, RNA processing site, effector binding site andstem-loop structure.

Degenerate Sequence: In this application degenerate sequences aredefined as populations of sequences where specific sequence positionsdiffer between different molecules or clones in the population. Thesequence differences may be a single nucleotide or multiple nucleotidesof any number, examples being 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40,50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000nucleotides, or any number in between. Sequence differences in adegenerate sequence may involve the presence of 2, 3 or 4 differentnucleotides in that position within the population of sequences,molecules or clones. Examples of degenerate nucleotides in a specificposition of a sequence are: A or C; A or G; A or T; C or G; C or T; G orT; A, C or G; A, C or T; A, G or T; C, G or T; A, C, G or T.

Discrete Random Polynucleotide: A discrete random polynucleotide refersto a specific polynucleotide within a mixed collection ofpolynucleotides, chosen randomly from the collection.

Expression: The term “expression”, as used herein, refers to thetranscription and stable accumulation of sense (mRNA) or antisense RNAderived from the nucleic acid disclosed, as well as the accumulation ofpolypeptide as a product of translation of mRNA.

Full-length Open Reading Frame: As used herein, a full-length openreading frame refers to an open reading frame encoding a full-lengthprotein which extends from its natural initiation codon to its naturalfinal amino-acid coding codon, as expressed in a cell or organism. Incases where a particular open reading frame sequence gives rise tomultiple distinct full-length proteins expressed within a cell or anorganism, each open reading frame within this sequence, encoding one ofthe multiple distinct proteins, can be considered full-length. Afull-length open reading frame can be continuous or may be interruptedby introns.

Fusion polynucleotide: A fusion polynucleotide as used in thisapplication refers to a polynucleotide that results from the operablejoining of two separate and distinct polynucleotides into a singlepolynucleotide. In the context of this application, the term in-framefusion polynucleotide is defined as a fusion polynucleotide encoding afusion polypeptide.

Fusion polypeptide: A fusion polypeptide is an expression productresulting from the fusion of two or more open reading frames thatoriginally coded for separate proteins.

Gene: The term “gene” refers to a nucleic acid fragment that is capableof being expressed as a specific protein, optionally includingregulatory sequences preceding (5′ non-coding sequences) and following(3′ non-coding sequences) the coding sequence. “Native gene” refers to agene as found in nature in its natural host organism. “Natural gene”refers to a gene complete with its natural control sequences such as apromoter and terminator. “Chimeric gene” refers to any gene thatcomprises regulatory and coding sequences that are not found together innature. Accordingly, a chimeric gene may comprise regulatory sequencesand coding sequences that are derived from different sources, orregulatory sequences and coding sequences derived from the same source,but arranged in a manner different than that found in nature. Similarly,a “foreign” gene refers to a gene not normally found in the hostorganism, but that is introduced into the host organism by genetransfer. Foreign genes can comprise native genes inserted into anon-native organism, or chimeric genes. A “transgene” is a gene that hasbeen introduced into the genome by a transformation procedure.

In-Frame: The term “in-frame” in this application, and particularly inthe phrase “in-frame fusion polynucleotide,” refers to the reading frameof codons in an upstream or 5′ polynucleotide or ORF as being the sameas the reading frame of codons in a polynucleotide or ORF placeddownstream or 3′ of the upstream polynucleotide or ORF that is fusedwith the upstream or 5′ polynucleotide or ORF. Such in-frame fusionpolynucleotides typically encode a fusion protein or fusion peptideencoded by both the 5′ polynucleotide and the 3′ polynucleotide.Collections of such in-frame fusion polynucleotides can vary in thepercentage of fusion polynucleotides that contain upstream anddownstream polynucleotides that are in-frame with respect to oneanother. The percentage in the total collection is at least 10% and cannumber 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%or any number in between.

Iterate/Iterative: In this application, to iterate means to apply amethod or procedure repeatedly to a material or sample. Typically, theprocessed, altered or modified material or sample produced from eachround of processing, alteration or modification is then used as thestarting material for the next round of processing, alteration ormodification. Iterative selection refers to a selection process thatiterates or repeats the selection two or more times, using the survivorsof one round of selection as starting material for the subsequentrounds.

Non-homologous: The term “non-homologous” in this application is definedas having sequence identity at the nucleotide level of less than 50%.

Open Reading Frame (ORF): An ORF is defined as any sequence ofnucleotides in a nucleic acid that encodes a protein or peptide as astring of codons in a specific reading frame. Within this specificreading frame, an ORF can contain any codon specifying an amino acid,but does not contain a stop codon. The ORFs in the starting collectionneed not start or end with any particular amino acid. The ORF may becontinuous or may be interrupted by introns.

Operably linked: The term “operably linked” refers to the association ofnucleic acid sequences on a single nucleic acid fragment so that thefunction of one is affected by the other. For example, a promoter isoperably linked with a coding sequence when it is capable of effectingthe expression of that coding sequence (i.e., that the coding sequenceis under the transcriptional control of the promoter). Coding sequencescan be operably linked to regulatory sequences in sense or antisenseorientation.

Percentage of sequence identity: The term “percent sequence identity”refers to the degree of identity between any given query sequence, e.g.SEQ ID NO: 102, and a subject sequence. A subject sequence typically hasa length that is from about 80 percent to 200 percent of the length ofthe query sequence, e.g., 80, 82, 85, 87, 89, 90, 93, 95, 97, 99, 100,105, 110, 115, or 120, 130, 140, 150, 160, 170, 180, 190 or 200 percentof the length of the query sequence. A percent identity for any subjectnucleic acid or polypeptide relative to a query nucleic acid orpolypeptide can be determined as follows. A query sequence (e.g. anucleic acid or amino acid sequence) is aligned to one or more subjectnucleic acid or amino acid sequences using the computer program ClustalW(version 1.83, default parameters), which allows alignments of nucleicacid or protein sequences to be carried out across their entire length(global alignment, Chenna 2003).

ClustalW calculates the best match between a query and one or moresubject sequences, and aligns them so that identities, similarities anddifferences can be determined. Gaps of one or more residues can beinserted into a query sequence, a subject sequence, or both, to maximizesequence alignments. For fast pairwise alignment of nucleic acidsequences, the following default parameters are used: word size: 2;window size: 4; scoring method: percentage; number of top diagonals: 4;and gap penalty: 5. For multiple alignment of nucleic acid sequences,the following parameters are used: gap opening penalty: 10.0; gapextension penalty: 5.0; and weight transitions: yes. For fast pairwisealignment of protein sequences, the following parameters are used: wordsize: 1; window size: 5; scoring method: percentage; number of topdiagonals: 5; gap penalty: 3. For multiple alignment of proteinsequences, the following parameters are used: weight matrix: blosum; gapopening penalty: 10.0; gap extension penalty: 0.05; hydrophilic gaps:on; hydrophilic residues: Gly, Pro, Ser, Asn, Asp, Gln, Glu, Arg, andLys; residue-specific gap penalties: on. The ClustalW output is asequence alignment that reflects the relationship between sequences.ClustalW can be run, for example, at the Baylor College of MedicineSearch Launcher website and at the European Bioinformatics Institutewebsite on the World Wide Web (ebi.ac.uk/clustalw).

To determine a percent identity of a subject or nucleic acid or aminoacid sequence to a query sequence, the sequences are aligned usingClustal W, the number of identical matches in the alignment is dividedby the query length, and the result is multiplied by 100. It is notedthat the percent identity value can be rounded to the nearest tenth. Forexample, 78.11, 78.12, 78.13, and 78.14 are rounded down to 78.1, while78.15, 78.16, 78.17, 78.18, and 78.19 are rounded up to 78.2.

Plasmid: The terms “plasmid” and “vector” refer to genetic elements usedfor carrying genes which are not a natural part of a cell or anorganism. Vectors can either integrate into the genome or can bemaintained extrachromosomally as linear or circular DNA fragments. Suchelements may be autonomously replicating sequences, genome integratingsequences, phage or nucleotide sequences, linear or circular, of asingle- or double-stranded DNA or RNA, derived from any source, in whicha number of nucleotide sequences have been joined or recombined into aunique construction which is useful for introducing polynucleotidesequences into a cell or an organism.

Phenotypic Value: Phenotypic value refers to a quantitative measure of aphenotype or a trait exhibited by an organism. For example, heightmeasured in feet is a phenotypic value corresponding to body height inhumans.

Promoter: The term “promoter” refers to a DNA sequence capable ofcontrolling the expression of a coding sequence or functional RNA. Ingeneral, a coding sequence is located 3′ to a promoter sequence.Promoters may be derived in their entirety from a native gene, or becomposed of different elements derived from different promoters found innature, or even comprise synthetic DNA segments. It is understood bythose skilled in the art that different promoters may direct theexpression of a gene in different tissues or cell types, or at differentstages of development, or in response to different environmental orphysiological conditions. Promoters which cause a gene to be expressedin most cell types at most times are commonly referred to as“constitutive promoters”. It is further recognized that since in mostcases the exact boundaries of regulatory sequences have not beencompletely defined, DNA fragments of different lengths may haveidentical promoter activity.

Random/Randomized: made or chosen without method or conscious decision.

Randomized In-frame Fusion Polynucleotides: As used herein, this phraserefers to polynucleotides in one or more starting populations fused toeach other in a random manner to form randomized fusion polynucleotides,each randomized fusion polynucleotide comprising two or more members ofthe starting population(s). The random nature of the fusion is such thatthe association between different polynucleotides capable of fusing isnot deliberately biased or directed, so that each startingpolynucleotide has an equal or similar probability to be represented inthe final population of fusion polynucleotides, and that it has an equalor similar probability to be fused with any other member of the startingpopulation(s).

Randomized Translational Fusion: A randomized translational fusion is aprocess by which polynucleotides are randomly fused in a manner that theORFs specified by the individual polynucleotide sequences are fusedin-frame, to result in a fusion polynucleotide that encodes a fusionprotein.

Randomly Fused: The term “randomly fused” refers to a process by which acollection of fused polynucleotides is generated from one or morecollections of starting polynucleotides, where each member of thestarting polynucleotide collection(s) has an equal or similarprobability of joining to each other member. The objective of generatingrandomly fused polynucleotides is typically to generate all possiblecombinations, or as many combinations as possible, of fused members orsequences.

Resistance to a compound or stress, as described herein, implies thatthe cell is able to grow and divide under unfavorable conditions thatwould normally be inhibitive to growth.

Stringency of selection: The term “stringency of selection” refers toselection intensity, or the degree to which selective conditions affectthe probability of an organism surviving the selection. A higherstringency of selection implies a higher selection intensity, with lowersurvival rates expected; a lower stringency of selection implies a lowerselection intensity, with higher survival rates expected. Survival of aparticular organism or population of organisms under selectionultimately depends on the fitness or viability of that organism orpopulation of organisms under the selective conditions

Tolerance of a compound or stress, as described herein, implies that thecell is able to survive under unfavorable conditions that would normallybe lethal to the cell.

Transformed: The term “transformed” means genetic modification byintroduction of a polynucleotide sequence.

Transformation: As used herein the term “transformation” refers to thetransfer of a nucleic acid fragment into a host organism, resulting ingenetically stable inheritance. Host organisms containing thetransformed nucleic acid fragments are referred to as “transgenic” or“recombinant” or “transformed” organisms.

Transformed Organism: A transformed organism is an organism that hasbeen genetically altered by introduction of a polynucleotide sequenceinto the organism's genome.

The term unfavorable conditions as used herein implies any part of thegrowth condition, physical or chemical, that results in slower growththan under normal growth conditions, or that reduces the viability ofcells compared to normal growth conditions.

The sequences of the 83 fusion polynucleotides are given in SEQ ID NO:1-63 and 205-224;the amino acid sequence of the proteins encoded by thefusion polynucleotides are provided in SEQ ID NO: 64-126 and 225-244.These fusion polynucleotides are useful for developing alcohol andstress resistance and tolerance traits in a target organism. In general,expression of one or more of the fusion polynucleotides described hereincan enhance stress and alcohol tolerance in a cell or organism.

Target organisms for the expression and use of the fusionpolynucleotides described herein include but are not limited to: baker'syeast Saccharomyces cerevisiae; other Saccharomyces species;Schizosaccharomyces pombe; Kluyveromyces species such as K. lactis, K.marxianus and K thermotolerans; Candida species such as C. albicans, C.glabrata, C. stellate, C. tropicalis, C. dubliniensis and C. keroseneae;Pichia species such as P. angusta, P. anomala, P. membranifaciens and P.pastoris; oleaginous yeasts such as Yarrowia hpolytica; other yeastspecies such as Dekkera/Brettanomyces species; Brettanomycesbruxellensis; Torulaspora delbrueckii; Zygosaccharomyces bailii; andother yeasts, fungi, microbial eukaryotes and eukaryotic algae.

The 83 fusion polynucleotides described herein confer resistance ortolerance to a variety of stresses and unfavorable conditions,including, but not limited to high temperature; low temperature; low pH;high pH; high salt concentration; high osmotic strength; low osmoticstrength; presence of oxidizing agents (hydrogen peroxide and otherinorganic peroxides; inorganic oxidizing agents such as potassiumpermanganate; organic peroxides and hydroperoxides such as ethylhydroperoxide, diacetyl peroxide, diethyl maleate, tert-butylhydroperoxide, cumyl hydroperoxide and ascaridole); high pressure; lowpressure; ionizing radiation such as ultraviolet, X-ray or gammairradiation; presence of toxic metal ions such as those derived from theelements Ag (silver), As (arsenic), Cd (cadmium), Cr (chromium), Co(cobalt), Cu (copper), Hg (mercury), Ni (nickel), Pb (lead), Pt(platinum), Sb (antimony), Se (selenium), Tl (thallium), or Zn (zinc);high concentrations of alcohols such as ethanol, propanol, iso-propanol,butanol, iso-butanol, alcohols with carbon chains exceeding 4 carbons;high concentrations of alkanes such as butane, pentane, hexane, heptane,octane, nonane, decane, undecane, dodecane, hexadecane, or isomersthereof; presence of furfural, 5-hydroxymethylfurfural, benzoic acidderivatives (for example p-hydroxybenzoic acid), and other toxic ligninbreakdown products found in sugar preparations derived from biomass (Luo2002); or unfavorable conditions or stresses caused by the presence ofany other toxic compound or combinations of compounds, or anycombination of the above.

In one embodiment, one or more of the fusion polynucleotides is combinedwith control elements such as a promoter and/or terminator in a mannerthat the promoter is upstream of the 5′ end of the fusionpolynucleotide's coding region and the terminator is downstream of the3′ end of the fusion polynucleotide' coding region. If the controlelements are selected from such sequences that are known or suspected tobe active in the target cell or organism, then the resulting expressionconstructs, or ‘expression cassettes’, each consisting of a promoter, afusion polynucleotide, and a terminator, confer expression of the fusionpolynucleotide when introduced into a cell.

Promoters used for expression of fusion polynucleotides may be strongpromoters that result in high levels of protein expression, or weakpromoters that result in low levels of protein expression, or promotersof intermediate strength. Promoters may also be constitutive, beingexpressed in all or most cells and in all or most stages of growth, orspecific promoters whose activity depends on specific growth states ormetabolic states. Inducible promoters, whose activity depends on thepresence of a specific chemical or metabolite or growth condition whichinduces the promoter to be active, or repressible promoters, which canbe shut off or reduced in activity in the presence of a specificchemical or metabolite or growth condition are also suitable. Forexample, a fusion polynucleotide encoding a fusion protein conferringstress tolerance can be expressed using a promoter that is induced underconditions of abiotic stress or in the presence of toxic andgrowth-inhibiting compounds in the growth medium.

Terminators used for expression of fusion polynucleotides may also varyin their activity. The function of a terminator in gene expression is incompleting the transcription process and influencing mRNA half-life.Expression cassettes of fusion polynucleotides may contain strong orweak terminators or terminators of intermediate activity that predisposean mRNA to high, low or intermediate levels of stability. Suchterminators are suitable for pairing with strong, weak or intermediatepromoters.

The fusion polynucleotide expression cassettes may be further combinedwith sequences useful for propagation, or for selection of cell clonesthat have taken up DNA introduced into a population of cells. Suchsequences may include, but are not limited to: centromeres, autonomouslyreplicating elements, 2 micron plasmid origins of replication,prototrophic marker genes such as the S. cerevisiae genes URA3, TRP1,HIS3 or LEU2, or marker genes conferring resistance to antibiotics suchas the kanamycin resistance gene conferring resistance to theaminoglycoside antibiotic G418 and related antibiotics.

Combinations of fusion polynucleotide expression cassettes and one ormore sequences useful for propagation in the target organism may beinserted into a plasmid vector and cloned in E. coli or another suitableorganism that allows propagation and replication of the joinedpolynucleotide sequences (Sambrook 1989). They may also be joined andthen amplified using in vitro amplification methods such as thepolymerase chain reaction.

Individual fusion polynucleotide expression cassettes combined withother sequences are then introduced into yeast in a manner that theybecome stably established as part of the yeast genome. There are avariety of suitable methods or introducing nucleic acids into yeast,including lithium acetate transformation (Gietz 2002, Gietz 2006, Gietz2007), electroporation, spheroplast transformation, biolistic particlebombardment, and a glass bead method (Gietz 2001, Kawai 2010). Othermethods of yeast transformation are also known, including use of 2micron plasmids, and integrative transformation where the fusionpolynucleotide is stably integrated within a yeast chromosome. InSaccharomyces cerevisiae, site-specific integration is routine and theintegration site is dependent on sequences homologous to chromosomalsites that are present on the integrating nucleic acid.

After introduction into yeast, the population of yeast cells exposed tonucleic acid comprising the polynucleotide expression cassettes many becultured in a medium allowing isolation of yeast clones that have takenup the polynucleotide expression cassettes and incorporated them intotheir genome. For example, if a fusion polynucleotide expressioncassette is joined to a URA3 selectable marker gene and introduced intoa ura-strain of yeast, then the resulting cell population, when platedon solid minimal media lacking uracil, will only allow growth ofcolonies that have stably taken up the URA3 selectable marker. If theURA3 selectable marker is covalently associated with yeast centromeresequences and a yeast autonomously replicating sequence as well as afusion polynucleotide expression cassette, then the resulting yeastcentromeric plasmid can become established in the yeast genome as acircular mini-chromosome, whose sequences are faithfully inheritedduring cell division.

In another embodiment, two or more fusion polynucleotides can beintroduced into the same cell or organism to cause synergistic effects,and to result in a cell or an organism with higher levels of stresstolerance than observed when using only a single fusion polynucleotide.Such combinations of fusion polynucleotides can also arise in theprocess of screening a cell or an organism transformed with one or morefusion polynucleotides for enhanced resistance to or tolerance ofstresses and toxic compounds by transforming this organism with alibrary of fusion polynucleotides and screening the resultingtransformants for higher levels of resistance or tolerance.

After delivery of a fusion polynucleotide and its control sequences intoa cell, and its selection for presence in the cell, a variety of testsfor resistance to and tolerance of stresses and toxic compounds areperformed, which typically include exposure of a cell population to anunfavorable condition for a specified period of time and monitoring ofthe cell population during or after exposure. Changes in growth rate orcell viability are measured by comparing transformed cells tountransformed control cells or cells transformed with a control nucleicacid, such as an empty expression vector or a gene known not to conferresistance or tolerance phenotypes.

The fusion polynucleotides described herein can be used with yeaststrains used for fermentation or the production of fuels or chemicals.Fermentation and fuel or chemical production often involve culturing thecells in the presence of compounds that inhibit growth, compounds thatreduce cell viability, or abiotic conditions including but not limitedto high temperature, high salt, low pH or high pH that also eitherinhibit growth or reduce cell viability or cell productivity orcombinations thereof.

The fusion polynucleotides are also useful for modifying industrialyeast strains used for the production of ethanol, other alcohols, or avariety of chemicals.

Among the fusion polynucleotides listed herein are 61 different fusionpolynucleotides containing the yeast full length WHI2 open readingframe. The WHI2 gene (Saul 1985, Mountain 1990, Mountain 1990a,Radcliffe 1997, Kaida 2002, Leadsham 2009, Mendl 2011, Müller 2011), hasbeen previously implicated in stress tolerance (Mendl 2011, Müller 2011)as well as a variety of other cellular processes including regulation ofcolony morphology, control of cell division and cell size, nutritionalsensing, and mitochondrial function.

Among the fusion polynucleotides listed herein are two fusionpolynucleotides (M21-A09, M24-B12; SEQ ID NO:31 and 44) and theircorresponding fusion proteins (SEQ ID NO:94 and 107) which representfusions of the WHI2 protein with phosphatases.

Among the fusion polynucleotides listed herein are 14 fusionpolynucleotides (Y1-38A, SEQ ID NO:15; M22-C05, SEQ ID NO: 35; M23-C03,SEQ ID NO:37; M27-B07, SEQ ID NO: 56; PP0219-223, SEQ ID NOS: 205-209;and PP0229-233, SEQ ID NOS: 215-219) and their corresponding fusionproteins (Y1-38A, SEQ ID NO:78; M22-C05, SEQ ID NO: 98; M23-C03, SEQ IDNO:100; M27-B07, SEQ ID NO: 119; ; PP0219-223, SEQ ID NOS: 225-229; andPP0229-233, SEQ ID NOS: 235-239) which represent fusions of the WHI2protein with members of the KEOPS complex, a conserved protein complexwith a role in telomere maintenance, but of largely unknown function(Bianchi 2006, Downey 2006).

Among the fusion polynucleotides listed herein are 11 fusionpolynucleotides (M21-C08, SEQ ID NO: 32; PP0224-228, SEQ ID NOS:210-214; and PP0234-238, SEQ ID NOS: 220-224) and their correspondingfusion proteins (M21-C08, SEQ ID NO: 95; PP0224-228, SEQ ID NOS:230-234; and PP0234-238, SEQ ID NOS: 240-244) which represent fusions ofthe WHI2 protein with members of the EGO complex, a vacuolarmembrane-associated protein complex with a role in activation ofmicroautophagy during exit from rapamycin-induced growth arrest(Dubouloz 2005, Gao 2006, Piper 2006).

The sequences of 83 fusion polynucleotides are given in SEQ ID NO: 1-63and 205-224; the sequences of the fusion proteins that they encode aregiven in SEQ ID NO: 64-126 and 225-244.

Table 1 shows a summary of all 83 fusion polynucleotides and theircomponent ORFs

TABLE 1 Yeast active fusion polynucleotides + component open readingframes (ORFs) Nucleic Fusion acid Protein 5′ 5′ 3′ 3′ polynucleotide SEQSEQ ORF ORF 5′ ORF ORF ORF 3′ ORF name ID ID ID name description ID namedescription Y1-5A 1 64 YDR24 Putative protein of YOR WHI2 Activator ofthe general 6W-A unknown function 043W stress response Y1-7A 2 65 YHR12ANS1 Putative protein of YOR WHI2 Activator of the general 6C unknownfunction 043W stress response Y1-9A 3 66 YOL02 MIM1 Mitochondrial YDLSTE7 Signal transducing MAP 6C outer membrane 159W kinase kinase proteinY1-13A 4 67 YDR48 PAC11 Dynein YOR WHI2 Activator of the general 8Cintermediate chain 043W stress response Y1-17A 5 68 YOR04 WHI2 Activatorof the YJL1 YJL18 Putative protein of 3W general stress 85C 5C unknownfunction response Y1-18A 6 69 YLR37 STP3 Zinc-finger protein YOR OST3Oligosaccharyltransferase 5W of unknown 085W gamma subunit functionY1-19A 7 70 YOR04 WHI2 Activator of the YFL0 YFL06 Y′ elementhelicase-like 3W general stress 66C 6C protein response Y1-20A 8 71YHL02 WSC4 ER membrane YOR WHI2 Activator of the general 8W proteininvolved in 043W stress response translocation Y1-21A 9 72 YOL05 PSH1 E3ubiquitin ligase YLR GIS3 Protein of unknown 4W 094C function Y1-23A 1073 YFL066 YFL06 Y′ element YOR WHI2 Activator of the general C 6Chelicase-like 043W stress response protein Y1-25A 11 74 YGR06 ERG2 C-4methyl sterol YOR WHI2 Activator of the general 0W 5 oxidase 043W stressresponse Y1-28A 12 75 YJL065 DLS1 ISW2 chromatin YOR WHI2 Activator ofthe general C accessibility 043W stress response complex subunit Y1-33A13 76 YLR09 GIS3 Protein of unknown YKL SBA1 Hsp90 family co- 4Cfunction 117W chaperone Y1-34B 14 77 YML06 TEM1 GTP-binding YOR WHI2Activator of the general 4C protein of the ras 043W stress responsesuperfamily Y1-38A 15 78 YML03 CGI12 Component of the YOR WHI2 Activatorof the general 6W 1 KEOPS protein 043W stress response complex Y1-39B 1679 YLR46 YLR4 Dubious open YOR WHI2 Activator of the general 6C-B 66C-Breading frame 043W stress response Y1-40A 17 80 YDL10 YDL1 Putativelipase; YOR WHI2 Activator of the general 9C 09C involved in lipid 043Wstress response metabolism Y1-43A 18 81 YLR15 YLR1 Putative protein ofYOR WHI2 Activator of the general 4C-G 54C-G unknown function 043Wstress response Y1-45A 19 82 YIR016 YIR01 Putative protein of YOR WHI2Activator of the general W 6W unknown function 043W stress responseY1-47A 20 83 YER01 SPC25 Kinetochore-assoc. YOR WHI2 Activator of thegeneral 8C Ndc80 complex 043W stress response component Y1-48A 21 84YML11 ATR1 Multidrug efflux YOR WHI2 Activator of the general 6W pump043W stress response Y1-49A 22 85 YLR09 GIS3 Protein of unknown YHR YHR2Putative helicase 4C function 219W 19W Y1-58B 23 86 YDR37 LSM6 Lsm (LikeSm) YBL SSA3 ATPase involved in 8C protein 075C protein folding, stressresponse Y1-58C 24 87 YDR46 MRPL Mitochondrial YGL MTO1 Mitochondrialprotein 2W 28 large subunit 236C ribosomal protein Y1-66C 25 88 YGL23YGL2 Putative protein of YLR SSQ1 Mitochondrial hsp70- 5W 35W unknownfunction 369W type molecular chaperone Y1-67B 26 89 YLL03 UBI4 Ubiquitinessential YBL YBL0 Non-essential protein of 9C for the cellular 081W 81Wunknown function stress response Y2-28A 27 90 YLR15 YLR1 Putativeprotein of YOL MAM Protein required for 4C-G 54C-G unknown function 060C3 mitochondrial morphology M21-A02 28 91 YOR04 WHI2 Activator of the YHRRPS4B 40S ribosomal subunit 3W general stress 203C protein responseM21-A03 29 92 YOR04 WHI2 Activator of the YLR GIS3 Protein of unknown 3Wgeneral stress 094C function response M21-A04 30 93 YGR20 TRX2Cytoplasmic YOR WHI2 Activator of the general 9C thioredoxin 043W stressresponse isoenzyme M21-A09 31 94 YGR20 YCH1 Phosphatase YOR WHI2Activator of the general 3W similar to Cdc25p 043W stress responseM21-C08 32 95 YBR07 SLM4 Component of the YOR WHI2 Activator of thegeneral 7C EGO protein 043W stress response complex M21-D06 33 96 YNL08SNN1 Putative protein of YOR WHI2 Activator of the general 6W unknownfunction 043W stress response M22-C01 34 97 YPR08 TEF1 Translational YORWHI2 Activator of the general 0W elongation factor 043W stress responseEF-1 alpha M22-C05 35 98 YKR09 PCC1 Component of the YOR WHI2 Activatorof the general 5W-A KEOPS protein 043W stress response complex M22-D0136 99 YIR015 RPR2 Subunit of nuclear YOR WHI2 Activator of the general WRNase P 043W stress response M23-C03 37 100 YJL184 GON7 Component of theYOR WHI2 Activator of the general W KEOPS protein 043W stress responsecomplex M23-D02 38 101 YPL250 ICY2 Protein of unknown YJL2 NCE1 Proteinof unknown C function 05C 01 function M23-D09 39 102 YMR22 YMR2 NADP(+)-YBR MSI1 Subunit of chromatin 6C 26C dependent 195C assembly factor Idehydrogenase M23-E02 40 103 YEL03 HYP2 Translation YOR WHI2 Activatorof the general 4W elongation factor 043W stress response eIF-5A M23-F0241 104 YPL250 ICY2 Protein of unknown YOR WHI2 Activator of the generalC function 043W stress response M23-H01 42 105 YLR15 YLR1 Putativeprotein of YGR SPT4 Pol I and Pol II 4C-G 54C-G unknown function 063Ctranscriptional regulator M24-A05 43 106 YNR04 MSO1 Secretory vesicleYOR WHI2 Activator of the general 9C docking complex 043W stressresponse component M24-B12 44 107 YMR15 TPP1 DNA 3′- YOR WHI2 Activatorof the general 6C phosphatase 043W stress response M24-D11 45 108 YBR19MSI1 Subunit of YOR RAS1 G-protein signaling 5C chromatin 101W GTPaseassembly factor I M24-E05 46 109 YGR20 YCH1 Phosphatase YLR GIS3 Proteinof unknown 3W similar to Cdc25p 094C function M24-F06 47 110 YHR05 CUP1-Metallothionein YOR WHI2 Activator of the general 5C 2 binding copperand 043W stress response cadmium M25-E1 48 111 YJR120 YJR12 Protein ofunknown YOR WHI2 Activator of the general W 0W function 043W stressresponse M25-F4 49 112 YHR05 CUP1- Metallothionein YOR WHI2 Activator ofthe general 5C 2 043W stress response M25-G8 50 113 YPR06 FCY1 CytosineYOR WHI2 Activator of the general 2W deaminase 043W stress responseM25-G10 51 114 YMR19 ICY1 Protein of unknown YBR MSI1 Subunit ofchromatin 5W function 195C assembly factor I M25-H11 52 115 YLR16 YLR1Putative protein of YOR WHI2 Activator of the general 2W 62W unknownfunction 043W stress response M26-A12 53 116 YMR19 ICY1 Protein ofunknown YOR WHI2 Activator of the general 5W function 043W stressresponse M26-D6 54 117 YNL25 ATX1 Cytosolic copper YOR WHI2 Activator ofthe general 9C metallochaperone 043W stress response M27-A1 55 118 YDR43NPL3 RNA-binding YOR WHI2 Activator of the general 2W protein 043Wstress response M27-B7 56 119 YOR04 WHI2 Activator of the YML CGI12Component of the 3W general stress 036W 1 KEOPS protein complex responseM27-F8 57 120 YDR24 YDR2 Putative protein of YHR SOD2 Mitochondrial 6W-A46W- unknown function 008C manganese superoxide A dismutase M28-A4 58121 YER01 SPC25 Kinetochore-assoc. YNL YNL0 Putative protein of 8C Ndc80complex 042W-B 42W-B unknown function component M28-C9 59 122 YDR24 YDR2Putative protein of YPL1 TGS1 Trimethyl guanosine 6W-A 46W-A unknownfunction 57W synthase M28-D6 60 123 YBR19 YBR1 Putative protein of YLRGIS3 Protein of unknown 7C 97C unknown function 094C function M28-E4 61124 YDR37 LSM6 Lsm (Like Sm) YLR GIS3 Protein of unknown 8C protein 094Cfunction M29-E7 62 125 YGR06 SPT4 Pol I and Pol II YOR WHI2 Activator ofthe general 3C transcriptional 043W stress response regulator M30-E11 63126 YLR04 PDC1 Pyruvate YIL0 BCY1 cAMP-dep protein 4C decarboxylase 33Ckinase regulatory subunit PP0219 205 225 YGR26 BUD3 Component of the YORWHI2 Activator of the general 2C 2 KEOPS protein 043W stress responsecomplex PP0220 206 226 YJL184 GON7 Component of the YOR WHI2 Activatorof the general W KEOPS protein 043W stress response complex PP0221 207227 YKR03 KAE1 Component of the YOR WHI2 Activator of the general 8CKEOPS protein 043W stress response complex PP0222 208 228 YKR09 PCC1Component of the YOR WHI2 Activator of the general 5W-A KEOPS protein043W stress response complex PP0223 229 209 YML03 CGI12 Component of theYOR WHI2 Activator of the general 6W 1 KEOPS protein 043W stressresponse complex PP0224 210 230 YBR07 SLM4 Component of the YOR WHI2Activator of the general 7C EGO protein 043W stress response complexPP0225 211 231 YGR16 GTR2 Component of the YOR WHI2 Activator of thegeneral 3W EGO protein 043W stress response complex PP0226 212 232 YKL14LTV1 Component of the YOR WHI2 Activator of the general 3W EGO protein043W stress response complex PP0227 213 233 YKR00 MEH1 Component of theYOR WHI2 Activator of the general 7W EGO protein 043W stress responsecomplex PP0228 214 234 YML12 GTR1 Component of the YOR WHI2 Activator ofthe general 1W EGO protein 043W stress response complex PP0229 215 235YOR04 WHI2 Activator of the YGR BUD3 Component of the 3W general stress262C 2 KEOPS protein complex response PP0230 216 236 YOR04 WHI2Activator of the YJL1 GON7 Component of the 3W general stress 84W KEOPSprotein complex response PP0231 217 237 YOR04 WHI2 Activator of the YKRKAE1 Component of the 3W general stress 038C KEOPS protein complexresponse PP0232 218 238 YOR04 WHI2 Activator of the YKR PCC1 Componentof the 3W general stress 095W-A KEOPS protein complex response PP0233219 239 YOR04 WHI2 Activator of the YML CGI12 Component of the 3Wgeneral stress 036W 1 KEOPS protein complex response PP0234 220 240YOR04 WHI2 Activator of the YBR SLM4 Component of the EGO 3W generalstress 077C protein complex response PP0235 221 241 YOR04 WHI2 Activatorof the YGR GTR2 Component of the EGO 3W general stress 163W proteincomplex response PP0236 222 242 YOR04 WHI2 Activator of the YKL LTV1Component of the EGO 3W general stress 143W protein complex responsePP0237 223 243 YOR04 WHI2 Activator of the YKR MEH1 Component of the EGO3W general stress 007W protein complex response PP0238 224 244 YOR04WHI2 Activator of the YML GTR1 Component of the 3W general stress 121WEG + A2:I85 response

EXAMPLES Example 1 Generation of Fusion Polynucleotides Encoding FusionPolypeptides

A yeast fusion polynucleotide library was generated according to themethod set forth in U.S. application Ser. No. 14/134,619, which ishereby incorporated by reference in its entirety.

Example 2 Resistance and Tolerance Phenotypes in Galactose-ContainingMedia of Yeast Strains Transformed with Fusion PolynucleotideCentromeric Plasmids

All fusion polynucleotides generated from Example 1 were cloned into aderivative of the yeast centromeric plasmid p416-GAL1 (Mumberg 1995,Funk 2002). The yeast centromeric plasmid p416-GAL1 contains thefollowing sequences for plasmid propagation in yeast and/or E. coli andexpression of an inserted polynucleotide: the bacterial replicon ofplasmid pMB1, the bacterial ampicillin-resistance gene, the yeastCEN6/ARSH4 cassette (Sikorski 1989) containing the chromosome 6centromere and the yeast histone H4-associated autonomously replicatingsequence (ARS), the yeast URA3 prototrophic marker gene, and the yeastGAL1 promoter and CYC1 terminator placed adjacent to each other in amanner that allows expression of coding regions inserted therebetween.The nucleotide sequence of the p416-GAL1 derivative is SEQ ID NO 127.All polynucleotides were cloned between nucleotide numbers 3206 and 3207of SEQ ID NO 127.

The resulting set of centromeric plasmids containing expressioncassettes of the 63 fusion polynucleotides under control of the S.cerevisiae GAL1 promoter (SEQ ID NO: 1-63) are hereafter referred to as‘fusion polynucleotide GAL1 centromeric plasmids’. Individual fusionpolynucleotide GAL1 centromeric plasmids are referred to by the SEQ IDnumber of the fusion polynucleotide contained therein, i.e. ‘fusionpolynucleotide GAL1 centromeric plasmid #5’ corresponds to SEQ ID NO: 5cloned between nucleotide numbers 3206 and 3207 of the p416-GAL1 vector(SEQ ID NO 127).

All experiments measuring tolerance and resistance activities of thefusion polynucleotides were performed using strain BY4741 (Brachmann1998) transformed with each fusion polynucleotide GAL1 centromericplasmid.

Yeast transformations were performed by the lithium acetate—heat shockmethod (Gietz 2002, Gietz 2006, Gietz 2007). The yeast strain BY4741(Brachmann 1998) from a plate or an overnight culture was inoculatedinto 50 ml of YPD medium (for 1 L medium, 20 g Bacto Peptone, 10 g BactoYeast Extract are combined and after autoclaving 20 g Glucose are addedas a 20% sterile solution) at 30° C. on a shaker at 225 rpm from astarting density of 5×10⁶ cells/ml (cell density determined with ahemocytometer), and grown over several hours to a final cell density of2×10⁷ cells/ml. The cells were harvested by centrifuging at 3000 g for 5min, were then resuspended in 25 ml of sterile deionized water, andcentrifuged again. Cells were resuspended in 1 ml of sterile water,transferred to a 1.5 ml microcentrifuge tube, centrifuged for 30 sec at3000 rpm and the supernatant aspirated. The cell pellet was thenresuspended in 0.4 ml of sterile deionized water. The cell suspensionwas combined with 3.26 ml of transformation mix (2.4 ml of 50% w/v PEG3350, 360 μl 1M Lithium acetate and 500 μl 10 mg/ml sheared, boiledsalmon sperm DNA) and mixed well. Aliquots of DNA (100 ng-1 μg) werepipetted into separate 1.5 ml microcentrifuge tubes and combined with380 μl of the cell suspension in transformation mix. The cell/DNAmixture was mixed thoroughly and incubated at 42° C. on a shaker at 250rpm for 40 minutes. The transformations were then centrifuged for 1minute at 3000 rpm in a microcentrifuge, the supernatant aspirated andeach cell aliquot resuspended in 0.5-1 ml sterile deionized water.Depending on the desired density of colonies, 10 μl to 1 ml of the cellsuspension were plated with sterile 4 mm glass beads onto one 10 cm or15 cm plate containing synthetic complete uracil dropout solid mediumhaving glucose as a carbon source (SCD-Ura agar; for 1 L, 6.7 g yeastnitrogen base, 0.77 g uracil dropout mix and 15 g Bacto agar arecombined, the pH is adjusted with 120 μ10N NaOH to bring the pH to5.6-5.8, and after autoclaving 20 g glucose are added as a 20% sterilesolution). After drying, the plates were covered and incubated at 30° C.for 3-4 days until colonies of transformants had formed. The individualfusion polynucleotide GAL1 centromeric plasmid transformants of strainBY4741 were maintained on SCD-Ura agar.

Resistance to and tolerance of the 64 yeast strains containing fusionpolynucleotides (63 fusion polynucleotide GAL1 centromeric plasmids andone control vector) to heat (42° C.), ethanol (15%), butanol (3%), salt(2M NaCl) and low pH (pH 3.0 in 0.2M sodium acetate) were measured intriplicate.

The 64 strains were first cultured for 16 hours at 30° C., 800 rpm witha 3 mm radius of gyration in 96-well plates (2 ml square wells) insynthetic complete uracil dropout medium containing glucose andgalactose as carbon sources (SCDGal-Ura; for 1 L of medium, 6.7 g yeastnitrogen base, 0.77 g uracil dropout mix, and 5 g glucose are combined,the pH is adjusted to 5.6-5.8 by the addition of 120 μ10N NaOH and afterautoclaving 5 g glucose and 15 g galactose are added as sterile 20%solutions). The cell density was then measured with a hemocytometer, andthe cells diluted in fresh 96-well plates (2 ml square wells) to a finalcell density of 1×10⁷ cells/ml in 1.25 ml YPGal (for 1 L of medium, 20 gBacto Peptone and 10 g Bacto Yeast Extract are combined, and afterautoclaving 20 g galactose are added as a sterile 20% solution). Thecells were cultured for an additional 5 hours.

The cell suspensions were then added in 0.25 ml aliquots to new 96-wellplates (2 ml square wells), each well containing 0.25 ml of YPGal mediumwith a 2× concentration of one selective agent. The selective agentsused were: NaCl at a final concentration of 2M; ethanol at a finalconcentration of 15%; n-butanol at a final concentration of 3%; orsodium acetate pH 3.0 at a final concentration of 0.2M. The sodiumacetate was prepared as a 1M solution of sodium acetate pH 3.0 by mixing2.74 ml glacial acetic acid with 0.11 g anhydrous sodium acetate in 50ml final volume and filter sterilized using a 0.2 micron filter. Formeasuring heat tolerance, the 0.25 ml aliquots of cell suspension werecombined with 0.25 ml of YPGal and cultured in 96-well plates (2 mlsquare wells) at 42° C. All selective cultures were incubated for 3days: those measuring heat tolerance at 42° C. and all others at 30° C.The cultures were then spotted undiluted and in 1:10 dilutions ontofresh 15 cm plates containing YPD solid medium (for 1 L medium, 20 gBacto Peptone, 10 g Bacto Yeast Extract and 15 g Bacto Agar arecombined, and after autoclaving 20 g glucose are added as a sterile 20%solution). Spotting was conducted using a Bel-Art 96-well replicatingtool (Bel-Art Products) that deposits spots of approximately 3 μl fromeach well onto the recipient plate. Dilutions were made in syntheticcomplete uracil dropout medium containing glucose as a carbon source(SCD-Ura; for 1 L of medium, 6.7 g yeast nitrogen base and 0.77 g uracildropout mix are combined, the pH is adjusted to 5.6-5.8 by the additionof 120 μl 10N NaOH and after autoclaving 20 g glucose are added as asterile 20% solution).

The spots were allowed to dry, the plates incubated at 30° C. for 1 dayto allow surviving cells to grow into cell spots or individual colonies.The cell density of surviving cells from each transformant for eachselective condition was scored on a relative scale from 0-3, 0 being nogrowth, 1 being slight growth, 2 being significant growth and 3 beingconfluent growth. Both the undiluted and diluted spots were taken intoaccount to generate the score. Triplicate measurements were made foreach condition and control.

To obtain a final activity score for each fusion polynucleotide GAL1centromeric plasmid and each selective condition, the triplicate scoresfor each selective condition were added, and the triplicate score forthe control plasmid subtracted. Final activity scores below zero werescored as zero. All scores are shown in Table 2 under the columnslabeled ‘Activity scores (rich medium)’.

Resistance and tolerance of the 64 yeast strains to ethanol and butanolwere also measured in minimal media containing raffinose and galactoseas carbon sources. Here, the 64 strains were first grown in 96 wellplates (2 ml square wells) containing minimal uracil dropout mediumcontaining 2% raffinose as a carbon source (SCRaf-Ura; for 1 L ofmedium, 6.7 g yeast nitrogen base and 0.77 g uracil dropout mix arecombined; pH is adjusted to 5.6-5.8 by the addition of 120 μl 10N NaOHand after autoclaving 20 g raffinose are added as a sterile 20%solution). Cells were incubated for 6 hours at 30° C. with constantshaking at 200 rpm. Expression of the fusion genes was then induced withgalactose at a final concentration of 2% and incubation continuedovernight. Subsequently, 0.1 OD cultures from each well were inoculatedinto minimal uracil dropout medium containing 1% raffinose and 2%galactose containing different concentrations of ethanol and butanol.Four concentrations each of ethanol (i.e. 8%, 11%, 14% and 17% v/v) andn-butanol (2%, 2.5%, 3% and 3.5% v/v) were used.

The 96-well culture plates (2 ml square wells) were covered withair-permeable sealing films and all plates were together further sealedin a large airtight plastic bag. This created a semi-aerobic conditionand the cultures were incubated in a shaking incubator at 30° C. for 3days. Using a Bel-Art 96-well replicating tool (Bel-Art Products) twodilutions of each culture (1:10 and 1:100) were spotted on minimaluracil dropout solid medium containing glucose as a carbon source(SCD-Ura agar; for 1 L of medium, 6.7 g yeast nitrogen base, 0.77 guracil dropout mix, and 15 g Bacto Agar are combined, the pH is adjustedto 5.6-5.8 by the addition of 120 μl 10N NaOH and after autoclaving 20 gglucose are added as a sterile 20% solution). The plates were incubatedat 30° C. for 2 days.

An image was taken of each plate and was scored for each dilution foreach fusion polynucleotide. A score of 0 to 5 was given to each spotbased on growth compared to the negative control strain. For each fusionpolynucleotide GAL1 centromeric plasmid and for each dilution, the scorewas multiplied by its corresponding concentration of butanol/ethanol andaveraged. The same scoring method was followed for the strain with thenegative control plasmid on the same plates: this provided thebackground. The final score was obtained by subtracting the averagescore of the negative control strain from the average score of thestrain with the individual fusion polynucleotide GAL1 centromericplasmids. All scores are shown in Table 2 under the columns labeled‘Activity scores (minimal medium)’.

Yeast strain BY4741, when transformed with each of the 63 fusionpolynucleotide GAL1 centromeric plasmids, showed significant resistanceto or tolerance of elevated temperature, ethanol, butanol, low pH and/orhigh salt compared to the negative control plasmid p416-GAL1, as shownin Table 2 below.

All resistance and tolerance scores above background are listed in Table2 below.

TABLE 2 Resistance and tolerance activities of 63 fusion polynucleotidesin galactose-containing rich medium in S. cerevisiae Fusion gene FusionActivity scores (rich medium) Activity scores nucleic gene pH 3 (minimalFusion acid protein (0.1M Salt medium) polynucleotide sequence sequenceHeat Ethanol Butanol sodium (2M Butanol Ethanol name SEQ ID SEQ ID (42°C.) (15%) (3%) acetate) NaCl) (3%) (15%) Y1-5A 1 64 3.75 0.00 4.75 2.754.75 0.0 35.0 Y1-7A 2 65 3.75 1.75 7.75 7.75 7.75 0.0 3.5 Y1-9A 3 660.00 1.75 4.75 4.75 4.75 0.0 0.8 Y1-13A 4 67 5.75 2.75 10.75 6.75 6.750.0 10.5 Y1-17A 5 68 6.75 2.75 7.75 8.75 7.75 0.0 7.0 Y1-18A 6 69 0.000.00 1.75 2.75 3.75 0.0 0.0 Y1-19A 7 70 0.75 5.75 7.75 8.75 7.75 0.0 9.8Y1-20A 8 71 6.75 0.00 3.75 2.75 2.75 0.0 23.8 Y1-21A 9 72 5.75 0.00 4.755.75 7.75 0.0 0.0 Y1-23A 10 73 6.75 0.00 4.75 5.75 4.75 0.0 0.8 Y1-25A11 74 0.75 0.00 1.75 2.75 1.75 0.0 7.0 Y1-28A 12 75 3.75 2.75 5.75 7.756.75 0.0 17.5 Y1-33A 13 76 6.75 0.00 0.00 1.75 4.75 0.0 0.0 Y1-34B 14 776.75 5.75 7.75 8.75 7.75 0.0 14.0 Y1-38A 15 78 6.75 2.75 7.75 8.75 4.750.0 7.0 Y1-39B 16 79 6.75 3.75 7.75 6.75 5.75 0.0 14.0 Y1-40A 17 80 5.750.00 1.75 5.75 4.75 0.0 7.0 Y1-43A 18 81 6.75 5.75 6.75 8.75 7.75 0.014.0 Y1-45A 19 82 6.75 0.00 0.00 0.00 1.75 0.0 0.0 Y1-47A 20 83 8.755.75 4.75 4.75 4.75 0.0 14.8 Y1-48A 21 84 6.75 5.75 4.75 8.75 7.75 0.010.5 Y1-49A 22 85 1.75 0.00 0.00 0.00 1.75 0.0 0.0 Y1-58B 23 86 0.000.00 0.00 0.00 0.00 0.0 0.0 Y1-58C 24 87 0.00 0.00 0.00 0.00 0.00 0.00.0 Y1-66C 25 88 0.00 0.00 0.00 0.00 0.00 0.0 0.0 Y1-67B 26 89 0.75 0.000.00 0.00 2.75 3.0 0.0 Y2-28A 27 90 0.75 0.00 0.00 0.00 0.00 0.0 0.0M21-A02 28 91 3.75 2.75 4.75 7.25 7.75 4.5 0.0 M21-A03 29 92 6.75 5.7510.75 11.75 7.75 16.0 0.0 M21-A04 30 93 6.75 0.00 4.75 5.75 4.75 4.5 0.0M21-A09 31 94 5.75 2.75 4.75 4.75 3.75 4.5 2.8 M21-C08 32 95 6.75 2.756.25 5.75 4.75 4.5 2.8 M21-D06 33 96 7.75 0.00 4.00 3.25 9.75 1.0 0.0M22-C01 34 97 6.75 3.00 7.00 9.25 9.75 16.4 0.0 M22-C05 35 98 6.75 3.008.00 6.25 6.75 2.5 0.0 M22-D01 36 99 4.75 3.00 7.00 6.25 9.75 3.0 0.0M23-C03 37 100 9.75 9.00 10.00 9.25 9.75 11.8 0.0 M23-D02 38 101 0.000.00 0.00 3.25 6.75 0.0 0.0 M23-D09 39 102 0.00 9.00 10.00 12.25 12.756.5 0.0 M23-E02 40 103 9.75 1.00 5.00 6.25 6.75 3.5 0.0 M23-F02 41 1049.75 9.00 10.00 12.25 12.75 1.6 0.8 M23-H01 42 105 2.75 0.00 3.00 6.256.75 0.0 0.0 M24-A05 43 106 0.75 3.00 7.00 6.25 9.75 3.5 13.0 M24-B12 44107 3.75 0.00 3.00 6.25 9.75 4.5 4.3 M24-D11 45 108 0.00 3.00 10.00 6.253.75 18.3 0.0 M24-E05 46 109 0.00 6.00 7.00 12.25 12.75 14.9 0.0 M24-F0647 110 6.75 6.00 7.00 9.25 9.75 7.8 0.0 M25-E1 48 111 9.75 3.00 4.006.25 9.75 4.0 0.0 M25-F4 49 112 9.75 0.00 0.00 6.25 6.75 2.5 0.0 M25-G850 113 9.75 0.00 1.00 6.25 6.75 2.5 0.0 M25-G10 51 114 1.75 6.00 1.008.25 8.75 0.0 0.0 M25-H11 52 115 6.75 0.00 0.00 1.25 4.75 2.5 0.0M26-A12 53 116 9.75 9.00 10.00 12.25 12.75 19.1 14.3 M26-D6 54 117 9.750.00 0.00 6.25 5.75 20.0 17.8 M27-A1 55 118 6.75 9.00 4.00 12.25 12.7520.0 21.3 M27-B7 56 119 9.75 0.00 1.00 6.25 6.75 5.6 0.0 M27-F8 57 1200.00 0.00 0.00 0.00 0.75 0.8 0.0 M28-A4 58 121 0.00 0.00 0.00 0.00 3.750.0 0.0 M28-C9 59 122 0.00 0.00 0.00 0.00 6.75 0.0 0.0 M28-D6 60 1233.75 0.00 4.00 6.25 9.75 2.5 0.0 M28-E4 61 124 3.75 3.00 4.00 9.25 12.750.0 0.0 M29-E7 62 125 6.75 3.00 7.00 9.25 7.75 4.4 7.0 M30-E11 63 1260.75 0.00 0.00 0.25 8.25 13.0 0.0 p416-GAL1 0.00 0.00 0.00 0.00 0.00 0.00.0

Example 3 Resistance and Tolerance Phenotypes in Glucose-ContainingMedia of Yeast Strains Transformed with Fusion PolynucleotideCentromeric Plasmids

63 fusion polynucleotides (SEQ ID NO: 1-63) were cloned into the yeastexpression vector p466-TEF1 (SEQ ID NO: 128), which is similar to theyeast centromeric plasmid p416-TEF1 (Mumberg 1995, Funk 2002). The yeastcentromeric plasmid p466-TEF1 contains the following sequences forplasmid propagation in yeast and E. coli, and expression of an insertedpolynucleotide: the bacterial replicon of plasmid pMB1; the bacterialampicillin-resistance gene; the yeast CEN6/ARSH4 cassette (Sikorski1989) containing the chromosome 6 centromere and the yeast histoneH4-associated autonomously replicating sequence (ARS); the yeast URA3prototrophic marker gene; and the yeast TEF1 promoter placed adjacent tothe CYC1 terminator in a manner that allows expression of coding regionsplaced between them. All polynucleotides were cloned between nucleotides3029 and 3030 of SEQ ID NO 128.

The resulting set of constructs are referred to as ‘fusionpolynucleotide TEF1 centromeric plasmids’. Each contains an expressioncassette of one of 63 fusion polynucleotides (SEQ ID NO: 1-63) undercontrol of the S. cerevisiae TEF1 promoter. All experiments measuringtolerance and resistance activities of the fusion polynucleotides wereperformed by using strain BY4741 (Brachmann 1998) that was transformedwith each fusion polynucleotide centromeric plasmid. Yeasttransformations were performed using the lithium acetate-heat shockmethod as described in Example 2. The individual fusion polynucleotidecentromeric plasmid transformants of strain BY4741 were maintained onminimal uracil dropout solid medium containing glucose as a carbonsource (SCD-Ura agar; for 1 L medium, 6.7 g yeast nitrogen base, 0.77 guracil dropout mix, and 15 g Bacto Agar are combined, the pH is adjustedto 5.6-5.8 with 120 μl 10N NaOH and after autoclaving 20 g of glucoseare added as a sterile 20% solution). Quantitative determinations ofethanol and butanol tolerance was conducted in rich medium containingglucose as a carbon source. Individual fusion polynucleotide centromericplasmids are referred to by the SEQ ID number of the fusionpolynucleotide contained therein, i.e. ‘fusion polynucleotide TEF1centromeric plasmid #5’ corresponds to SEQ ID NO 5 cloned betweennucleotide numbers 3029 and 3030 of the p466-TEF1 vector.

Resistance and tolerance of the 64 yeast strains (63 harboring thefusion polynucleotide constructs plus one strain with the controlvector) were tested for resistance and tolerance to heat (42° C.),ethanol (15%), butanol (3%) and low pH (pH 3.0 in 0.2M sodium acetate).Measurements were taken in quadruplicate; that is, two independenttransformants of each fusion polynucleotide centromeric plasmids wereeach tested in duplicate. Here, the 63 fusion polynucleotide strains andone control strain were first cultured in 96-well plates (2 ml squarewells) having 1 ml per well of synthetic complete uracil dropout mediumcontaining glucose as a carbon source (SCD-Ura: for 1 L of medium, 6.7 gyeast nitrogen base and 0.77 g uracil dropout mix are combined, the pHis adjusted to 5.6-5.8 by the addition of 120 μl 10N NaOH, and afterautoclaving 20 g glucose are added as a sterile 20% solution). Cellswere incubated for 16 hours at 30° C. with shaking at 800 rpm with a 3mm radius of gyration.

The cell densities were measured with a hemocytometer, the cellsuspensions diluted in a fresh 96-well plate (2 ml square wells) to afinal cell density of 1×10⁷ cells/ml in 1.25 ml SCD-Ura, and then grownfor an additional 2 hours under the same conditions. The cellsuspensions were then added in 0.25 ml aliquots to fresh 96-well plates(2 ml square wells) and each aliquot combined with 0.25 ml YPD medium(for 1 L of medium, 20 g Bacto Peptone and 10 g Bacto Yeast Extract arecombined, and after autoclaving 20 g glucose are added as a sterile 20%solution) containing a 2× concentration of one selective agent. Theselective agents used were: ethanol at a final concentration of 15%;n-butanol at a final concentration of 3%, or sodium acetate pH 3.0 at afinal concentration of 0.2M. The sodium acetate was prepared as a 1Msolution by mixing 2.74 ml glacial acetic acid with 0.11 g anhydroussodium acetate in 50 ml final volume and filter sterilized using a 0.2micron filter. For measuring heat tolerance, the 0.25 ml aliquots ofcell suspension were combined with 0.25 ml of SCD-Ura medium andcultured in 96-well plates (2 ml square wells) at 42° C. All selectivecultures were incubated for 48 hours; those measuring heat tolerance at42° C. and all others at 30° C.

Using a Bel-Art 96-well replicating tool (Bel-Art Products) the cultureswere then spotted undiluted or as a 1:10 dilution (diluted in SCD-Ura)onto fresh 15 cm plates containing YPD solid medium (for 1 L of medium,20 g Bacto Peptone, 10 g Bacto Yeast Extract and 15 g Bacto Agar arecombined, and after autoclaving 20 g glucose are added as a sterile 20%solution). Approximately 3 μl from each well was deposited onto therecipient plate. The spots were allowed to dry and the plates wereincubated at 30° C. for 1 day to allow surviving cells to grow into cellspots or individual colonies. The cell density of each transformant foreach selective condition was scored on a relative scale from 0-3; 0being no growth, 1 slight growth, 2 significant growth, and 3 confluentgrowth. Both the undiluted and diluted spots were taken into account togenerate the score. To obtain a final activity score for each fusionpolynucleotide TEF1 centromeric plasmid under each selective condition,the quadruplicate scores for each selective condition obtained for thecontrol plasmid were summed and then subtracted from the sum of thescores obtained with the fusion polynucleotide TEF1 centromericplasmids. Final activity scores below zero were scored as zero. Acumulative score was calculated by adding the individual activity scoresfor each fusion polunucleotide TEF1 centromeric plasmid. All scores areshown in Table 3 under the columns labeled ‘Activity scores’.

Yeast strain BY4741 transformed with the set of 63 fusion polunucleotideTEF1 centromeric plasmids showed significant resistance to or toleranceof elevated temperature, ethanol, butanol, and/or low pH compared to thenegative control plasmid p466-TEF1, as shown in Table 3 below.

Activity scores above background of the 63 fusion polynucleotide TEF1centromeric plasmids are shown in Table 3 below.

TABLE 3 Resistance and tolerance activities of 63 fusion polynucleotidesin glucose- containing rich medium in S. cerevisiae Fusion Activityscores polynucleotide Fusion pH 3 Fusion nucleic polynucleotide (0.1Mpolynucleotide acid protein Heat Ethanol Butanol sodium Cumulative nameSEQ ID SEQ ID (42° C.) (15%) (3%) acetate) score Y1-5A 1 64 1.5 4.5 3.06.0 15.0 Y1-7A 2 65 0.0 2.0 1.0 4.0 7.0 Y1-9A 3 66 0.0 0.0 0.0 0.0 0.0Y1-13A 4 67 1.0 5.0 3.0 6.0 15.0 Y1-17A 5 68 0.0 3.0 1.0 4.0 8.0 Y1-18A6 69 0.0 0.0 0.0 0.0 0.0 Y1-19A 7 70 2.0 4.0 3.0 6.0 15.0 Y1-20A 8 710.0 4.0 1.0 5.0 10.0 Y1-21A 9 72 0.0 1.0 1.0 2.0 4.0 Y1-23A 10 73 0.02.0 0.0 4.0 6.0 Y1-25A 11 74 0.0 0.0 0.0 0.0 0.0 Y1-28A 12 75 0.0 4.01.5 6.0 11.5 Y1-33A 13 76 0.0 0.0 0.0 0.5 0.5 Y1-34B 14 77 1.0 4.0 1.55.5 12.0 Y1-38A 15 78 1.0 4.0 1.0 6.0 12.0 Y1-39B 16 79 1.0 4.0 2.0 6.013.0 Y1-40A 17 80 1.0 4.0 2.0 6.0 13.0 Y1-43A 18 81 2.5 4.0 2.5 6.0 15.0Y1-45A 19 82 0.0 2.0 1.0 4.0 7.0 Y1-47A 20 83 1.0 3.0 1.0 6.0 11.0Y1-48A 21 84 0.0 3.0 1.0 6.0 10.0 Y1-49A 22 85 0.0 0.0 0.0 0.0 0.0Y1-58B 23 86 0.0 0.0 0.0 0.5 0.5 Y1-58C 24 87 0.0 0.0 0.0 0.0 0.0 Y1-6625 88 0.0 0.0 0.0 0.0 0.0 Y1-67B 26 89 0.0 0.0 0.0 0.0 0.0 Y2-28A 27 900.0 0.0 0.0 0.0 0.0 M21-A02 28 91 2.0 4.0 2.0 6.0 14.0 M21-A03 29 92 5.05.5 5.0 6.0 21.5 M21-A04 30 93 2.0 4.5 2.5 6.0 15.0 M21-A09 31 94 1.53.5 2.0 5.5 12.5 M21-C08 32 95 3.0 6.0 2.0 7.0 18.0 M21-D06 33 96 0.04.0 3.0 6.0 13.0 M22-C01 34 97 0.0 6.0 5.0 6.0 17.0 M22-C05 35 98 0.04.0 3.0 6.0 13.0 M22-D01 36 99 2.0 4.0 3.0 6.0 15.0 M23-C03 37 100 4.06.0 6.0 6.0 22.0 M23-D02 38 101 0.0 1.0 0.5 2.5 4.0 M23-D09 39 102 0.55.0 2.0 6.0 13.5 M23-E02 40 103 0.0 5.0 3.0 6.0 14.0 M23-F02 41 104 3.56.0 4.0 6.0 19.5 M23-H01 42 105 0.0 0.0 0.0 0.0 0.0 M24-A05 43 106 2.05.0 4.0 6.0 17.0 M24-B12 44 107 0.5 4.0 3.5 6.0 14.0 M24-D11 45 108 0.00.0 0.0 0.0 0.0 M24-E05 46 109 0.0 3.0 3.0 4.0 10.0 M24-F06 47 110 2.06.0 5.0 6.0 19.0 M25-E01 48 111 0.0 3.0 1.0 4.0 8.0 M25-F04 49 112 0.04.0 2.0 6.0 12.0 M25-G08 50 113 0.0 4.0 3.0 6.0 13.0 M25-G10 51 114 0.01.0 0.0 2.0 3.0 M25-H11 52 115 0.0 1.5 0.0 3.0 4.5 M26-A12 53 116 0.00.0 0.0 0.0 0.0 M26-D06 54 117 0.0 4.0 2.0 6.0 12.0 M27-A01 55 118 0.52.0 2.0 4.0 8.5 M27-B07 56 119 0.5 5.5 4.5 6.0 16.5 M27-F08 57 120 0.00.0 0.0 0.0 0.0 M28-A04 58 121 0.0 0.0 0.0 0.0 0.0 M28-C09 59 122 0.00.0 0.0 0.0 0.0 M28-D06 60 123 0.0 3.0 3.0 5.5 11.5 M28-E04 61 124 0.03.0 3.5 5.0 11.5 M29-E07 62 125 0.5 6.0 5.0 6.0 17.5 M30-E11 63 126 0.00.0 1.0 0.0 1.0 p466-TEF1 — — 0.0 0.0 0.0 0.0 0.0

Example 4 Quantitative Resistance and Tolerance Phenotypes inGalactose-Containing Media of Yeast Strains Transformed with FusionPolynucleotide Centromeric Plasmids

Eighteen (18) selected fusion polynucleotide GAL1 centromeric plasmidsfrom Example 2 were used to generate quantitative measurements ofethanol and butanol resistance and tolerance activity.

All experiments measuring tolerance and resistance activities wereperformed by using strain BY4741 (Brachmann 1998) transformed with eachfusion polynucleotide GAL1 centromeric plasmid. Yeast transformationswere performed using the lithium acetate-heat shock method as describedin Example 2. Four independent transformants of each fusionpolynucleotide GAL1 centromeric plasmid were tested. The individualfusion polynucleotide GAL1 centromeric plasmid transformants of strainBY4741 were maintained on minimal uracil dropout solid medium containingglucose as a carbon source (SCD-Ura agar; for 1 L medium, 6.7 g yeastnitrogen base, 0.77 g uracil dropout mix, and 15 g Bacto Agar arecombined, the pH is adjusted to 5.6-5.8 with 120 μl 10N NaOH and afterautoclaving 20 g of glucose are added as a sterile 20% solution).

Yeast strains transformed with the 18 fusion polynucleotide GAL1centromeric plasmids, as well as the p416-GAL1 control plasmid, weretested for resistance and tolerance to ethanol and butanol in minimalmedia containing raffinose and galactose as carbon sources. The strainswere first grown in 96-well plates (2 ml square wells) containingminimal uracil dropout medium containing 2% raffinose as a carbon source(SCRaf-Ura: for 1 L of medium, 6.7 g yeast nitrogen base and 0.77 guracil dropout mix are combined, the pH is adjusted to 5.6-5.8 by theaddition of 120 μl 10N NaOH and after autoclaving 20 g raffinose areadded as a sterile 20% solution). Cells were incubated for 16 hours at30° C. with constant shaking at 800 rpm.

The cell densities were then measured with a hemocytometer, and the cellsuspensions diluted to a final cell density of 1×10⁷ cells/ml in 1.25rich medium containing raffinose and galactose as carbon sources(YPRaf-Gal; for 1 L of medium: 20 g Bacto Peptone and 10 g Bacto YeastExtract are combined, and after autoclaving 20 g of raffinose and 20 gof galactose are added as sterile 20% solutions). Cells were culturedfor an additional 4 hours at 30° C. with constant shaking at 800 rpm.

The cell suspensions were then added in 0.25 ml aliquots to fresh96-well plates (2 ml square wells) and combined with 0.25 ml per well offresh YPRaf-Gal containing a 2× concentration of one selective agent.The selective agents used were: ethanol at a final concentration of 15%and n-butanol at a final concentration of 3%. All selective cultureswere incubated for 2 days at 30° C. with constant shaking at 800 rpm.

The cultures were then diluted 1:10 and 1:100 in minimal uracil dropoutmedium containing glucose as a carbon source (SCD-Ura; for 1 L medium,6.7 g yeast nitrogen base and 0.77 g uracil dropout mix are combined,the pH is adjusted to 5.6-5.8 by the addition of 120 μl 10N NaOH andafter autoclaving 20 g of glucose are added as a sterile 20% solution).100 μl aliquots of the undiluted and diluted cells were plated onto 10cm plates containing solid minimal uracil dropout medium containingglucose as a carbon source (SCD-Ura agar; for 1 L medium, 6.7 g yeastnitrogen base, 0.77 g uracil dropout mix and 20 g Bacto Peptone arecombined, the pH is adjusted to 5.6-5.8 by the addition of 120 μl 10NNaOH and after autoclaving 20 g of glucose are added as a sterile 20%solution). The plates were allowed to dry and were incubated at 30° C.for 2 days to allow surviving cells to grow into colonies. Colonies werecounted on each plate, and the average number of surviving cells and thestandard deviation (Std Dev) for each set of transformants werecomputed. The results are shown in Table 4 under the column labeled‘Number of surviving cells’. The number of starting cells in eachculture was approximately 2.5×10⁶.

From the numbers of surviving cells shown in Table 4 it is clear thatyeast strain BY4741 transformed with the 18 fusion polynucleotide GAL1centromeric plasmids showed significant resistance to or tolerance ofethanol and butanol, compared to the negative control plasmid p466-GAL1.

All surviving cell numbers are listed in Table 4 below.

TABLE 4 Butanol and ethanol resistance and tolerance activities of 18fusion polynucleotides in galactose-containing rich medium in S.cerevisiae Fusion polynucleotide Fusion Fusion nucleic polynucleotideNumber of surviving cells polynucleotide acid protein 3% butanol 15%ethanol name SEQ ID SEQ ID Mean Std Dev. Mean Std Dev. Y1-9A 3 66 3,525222 52,500 6,245 Y1-17A 5 68 9,600 294 165,500 21,315 Y1-20A 8 71 14,925704 100,500 8,426 Y1-21A 9 72 4,550 289 4,475 834 Y1-23A 10 73 8,525 45716,025 1,090 Y1-28A 12 75 7,400 455 26,400 7,189 Y1-33A 13 76 1,900 3929,575 1,962 Y1-34B 14 77 19,300 883 17,025 954 Y1-48A 21 84 7,800 44017,425 2,645 Y1-66C 25 88 1,875 330 18,675 2,012 M21-A09 31 94 17,500606 28,700 2,706 M23-D02 38 101 1,575 403 15,100 1,158 M23-H01 42 1052,350 129 650 129 M24-E05 46 109 9,225 1,195 14,725 1,480 M25-E01 48 11112,550 480 60,250 17,727 M26-A12 53 116 188,750 7,890 382,750 20,982M26-D06 54 117 13,625 377 7,650 1,287 M27-A01 55 118 19,250 656 21,6251,834 p416-GAL1 127 — 375 126 575 222

Example 5 Comparison of Resistance and Tolerance Phenotypes Conferred byFusion Polynucleotides and Individual ORFs in Glucose-Containing Medium

Nineteen (19) selected fusion polynucleotide TEF1 centromeric plasmidsfrom example 3 were used to generate quantitative measurements of heat,ethanol, butanol and low pH resistance and tolerance activity. The 19fusion polynucleotides were compared in their activity to the 19individual full length open reading frames (ORFs) that were the buildingblocks of the fusion polynucleotides.

The 19 fusion polynucleotides and the 19 individual ORFs were clonedinto the yeast expression vector p466-TEF1 (SEQ. ID No 128). The yeastcentromeric plasmid p466-TEF1 contains the following sequences forplasmid propagation in yeast and E. coli, and for expression of aninserted polynucleotide: the bacterial replicon of plasmid pMB1; thebacterial ampicillin-resistance gene; the yeast CEN6/ARSH4 cassette(Sikorski 1989) which contains the chromosome 6 centromere as well asthe yeast histone H4-associated autonomously replicating sequence (ARS);the yeast URA3 prototrophic marker gene; and the yeast TEF1 promoterplaced adjacent the CYC1 terminator in a manner that allows expressionof coding regions placed between them (see SEQ ID NO:128). Allpolynucleotides were cloned between nucleotides 3029 and 3030 of SEQ IDNO 128. The 19 fusion polynucleotides cloned into p466-TEF1 are referredto as ‘fusion polynucleotide TEF1 centromeric plasmids’ and the 19individual full length open reading frames cloned into p466-TEF1 arereferred to as ‘individual ORF TEF1 centromeric plasmids’, andcollectively as ‘TEF1 centromeric plasmids’ below.

All experiments measuring tolerance and resistance activities wereperformed using strain BY4741 (Brachmann 1998) that was transformed witheach of the 19 fusion polynucleotide TEF1 centromeric plasmids and the19 individual ORF TEF1 centromeric plasmids, as well as the p466-TEF1control vector. Yeast transformations were performed using the lithiumacetate-heat shock method as described in Example 2. Four independenttransformants of each TEF1 centromeric plasmid were tested. The BY4741transformants were maintained on minimal uracil dropout solid mediumcontaining glucose as a carbon source (SCD-Ura agar; for 1 L medium, 6.7g yeast nitrogen base, 0.77 g uracil dropout mix, and 15 g Bacto Agarare combined, the pH is adjusted to 5.6-5.8 with 120 μ10N NaOH and afterautoclaving 20 g of glucose are added as a sterile 20% solution).

Resistance and tolerance of the 39 transformed yeast strains to heat(42° C.), ethanol (15%), butanol (3%) and low pH (pH 3.0 in 0.2M sodiumacetate) were measured in quadruplicate by testing four independenttransformants of each construct. The 39 strains were first cultured in96-well plates (2 ml square wells) in 1 ml per well of syntheticcomplete uracil dropout medium containing glucose as a carbon source(SCD-Ura; for 1 L medium, 6.7 g yeast nitrogen base, and 0.77 g uracildropout mix are combined, the pH is adjusted to 5.6-5.8 by the additionof 120 μ10N NaOH and after autoclaving 20 g of glucose are added as asterile 20% solution). Cells were incubated for 16 hours at 30° C.shaking at 800 rpm with a 3 mm radius of gyration.

The cell densities were measured with a hemocytometer, the cellsuspensions diluted in a fresh 96-well plate (2 ml square wells) to afinal cell density of 1×10⁷ cells/ml in 1.25 ml SCD-Ura, and then grownfor an additional 2 hours. The cell suspensions were then added in 0.25ml aliquots to fresh 96-well plates (2 ml square wells) and combinedwith 0.25 ml per well of fresh SCD-Ura medium containing 2×concentration of one selective agent. The selective agents used were:ethanol at a final concentration of 15%, n-butanol at a finalconcentration of 3%, or sodium acetate pH 3.0 at a final concentrationof 0.2M. The sodium acetate was prepared as a 1M solution of sodiumacetate pH 3.0 by mixing 2.74 ml glacial acetic acid with 0.11 ganhydrous sodium acetate in a final volume of 50 ml and filtersterilized using a 0.2 micron filter. For measuring heat tolerance, the0.25 ml aliquots of cell suspension were combined with 0.25 ml ofSCD-Ura medium and cultured in 96-well plates (2 ml square wells) at 42°C. All selective cultures were cultured for 48 hours with constantshaking at 800 rpm; those measuring heat tolerance were incubated at 42°C. and all others at 30° C.

The cultures were then diluted 1:100 and 1:500 in SCD-Ura. 150 μlaliquots of the diluted cells were plated onto 10 cm plates containingsolid minimal uracil dropout medium containing glucose as a carbonsource (SCD-Ura agar; for 1 L medium, 6.7 g yeast nitrogen base, 0.77 guracil dropout mix, and 15 g Bacto Agar are combined, the pH is adjustedto 5.6-5.8 using 120 μl 10N NaOH, and after autoclaving 20g glucose areadded as a sterile 20% solution). The plates were allowed to dry andwere incubated at 30° C. for 2 days to allow surviving cells to growinto colonies. Colonies were counted on each plate, and the averagenumber of surviving cells and the standard deviations (Std Dev) fromeach set of transformants computed. Results are shown in Table 5 (FIGS.1 and 2) under the columns labeled ‘Number of surviving cells’. Thenumber of starting cells in each culture was approximately 2.5×10⁶.

Yeast strain BY4741 transformed with various fusion polynucleotide TEF1centromeric plasmids showed enhanced resistance to or tolerance of heat,ethanol, butanol and low pH, compared to the individual ORF TEF1centromeric plasmids, as shown in Table 5. Yeast strain BY4741containing the negative control plasmid p466-TEF1 (SEQ ID NO 128) servedas a control.

Only one of the individual ORFs (WHI2) showed any stress toleranceactivity; all the others had no measurable activity. Activities of thefusion polynucleotides that surpass the individual WHI2 ORF score areidentified by grey shaded cells in Table 5 (FIGS. 1 and 2). In manycases, the activity level is significantly above that of the individualWHI2 ORF, suggesting unexpected gain-of-function activity by the fusionpolynucleotides.

All resistance and tolerance scores are listed in Table 5 which is splitinto two parts; part A (FIG. 1) shows cell counts from heat and ethanolselections and part B (FIG. 2) shows cell counts from butanol and low pHselections.

Example 6 Comparison of Resistance and Tolerance Phenotypes Conferred byKEOPS/EGO Fusion Polynucleotides and Individual ORFs inGlucose-Containing Medium

Examples 2, 3 and 5 contain evidence that members of the yeast KEOPS andEGO protein complexes, when fused with the yeast WHI2 ORF, result inelevated levels of stress and alcohol tolerance in yeast strainsexpressing these fusion proteins (see data for fusion polynucleotidesY1-38A, M22-C05, M23-C03, M27-B07, and M21-C08 in Tables 2, 3, and 5.

Both the KEOPS complex and the EGO complex contain 5 different proteins.The members of the KEOPS complex are named BUD32, GON7, KAE1, PCC1 andCGI121. The members of the EGO complex are named SLM4, LTV1, MEH1, GTR1and GTR2.

To test whether all members of the KEOPS and EGO complexes are able toconfer stress and alcohol tolerance when fused with WHI2, 20 new fusionswere constructed that combined all 10 members of the two complexes withWHI2 in both orientations. That is, each complex member was present inthe fusion polypeptide in the 5′ position relative to WHI2 as well as inthe 3′ position. The 20 resulting fusion polynucleotides (PP0219-PP0238;SEQ ID NOs 205-224, respectively, whose protein sequences are given inSEQ ID NOs 225-244, respectively) were cloned into the yeast expressionvector p466-TEF1 (SEQ. ID No 128); see example 3 for details. Theresulting 20 new fusion polynucleotide constructs are referred to as the‘KEOPS/EGO TEF1 fusion polynucleotide centromeric plasmids’. Each of the11 full-length ORFs (BUD32, GON7, KAE1, PCC1,CGI121, SLM4, LTV1, MEH1,GTR1, GTR2 and WHI2) were also individually cloned into yeast expressionvector p466-TEF1 (SEQ. ID No 128); see example 3 for details. Theresulting 11 new individual full length ORF constructs are referred toas the ‘KEOPS/EGO TEF1 individual ORF centromeric plasmids’. Allcentromeric expression constructs discussed in this example arecollectively referred to as ‘TEF1 centromeric plasmids’.

The 20 KEOPS/EGO TEF1 fusion polynucleotide centromeric plasmids and the11 KEOPS/EGO TEF1 individual ORF centromeric plasmids were used togenerate quantitative measurements of heat, ethanol, butanol and low pHresistance/tolerance activity. The 20 fusion polynucleotides werecompared in their activity to the 11 individual ORFs that are thebuilding blocks of the 20 fusion polynucleotides.

All experiments measuring tolerance and resistance activities wereperformed using strain BY4741 (Brachmann 1998) transformed with each ofthe 20 KEOPS/EGO TEF1 fusion polynucleotide centromeric plasmids and the11 KEOPS/EGO TEF1 individual ORF centromeric plasmids, as well as thep466-TEF1 control vector. Yeast transformations were performed using thelithium acetate-heat shock method as described in Example 2. Fourindependent transformants of each TEF1 centromeric plasmid were tested.The BY4741 transformants were maintained on minimal uracil dropout solidmedium containing glucose as a carbon source (SCD-Ura agar; for 1 Lmedium, 6.7 g yeast nitrogen base, 0.77 g uracil dropout mix, and 15 gBacto Agar are combined, the pH is adjusted to 5.6-5.8 with 120 μl 10NNaOH and after autoclaving 20 g of glucose are added as a sterile 20%solution).

Resistance and tolerance of the 32 yeast strains (BY4741 transformedwith 20 KEOPS/EGO TEF1 fusion polynucleotide centromeric plasmids, 11KEOPS/EGO TEF1 individual ORF centromeric plasmids and one controlvector) to heat (42° C.), ethanol (15%), butanol (3%) and low pH (pH 3.0in 0.2M sodium acetate) were measured in quadruplicate by testing fourindependent transformants of each construct. The 32 strains were firstcultured in 96-well plates (2 ml square wells) in 1 ml per well ofsynthetic complete uracil dropout medium containing glucose as a carbonsource (SCD-Ura; for 1 L medium, 6.7 g yeast nitrogen base and 0.77 guracil dropout mix are combined, the pH is adjusted to 5.6-5.8 with 120μl 10N NaOH, and after autoclaving 20 g of glucose are added as asterile 20% solution). The cells were incubated for 16 hours at 30° C.with constant shaking at 800 rpm with a 3 mm radius of gyration.

The cell densities were measured with a hemocytometer, the cellsuspensions diluted in a fresh 96-well plate (2 ml square wells) to afinal cell density of 1×10⁷ cells/ml in 1.25 ml SCD-Ura, and then grownfor an additional 2 hours. The cell suspensions were added in 0.25 mlaliquots to fresh 96-well plates (2 ml square wells) and combined with0.25 ml per well of fresh SCD-Ura medium containing a 2× concentrationof one selective agent. The selective agents used were: ethanol at afinal concentration of 15%; n-butanol at a final concentration ofn-butanol or sodium acetate pH 3.0 at a final concentration of 0.2M. Thesodium acetate was prepared as a 1M solution of sodium acetate pH 3.0 bymixing 2.74 ml glacial acetic acid with 0.11 g anhydrous sodium acetatein 50 ml final volume and filter sterilized using a 0.2 micron filter.For measuring heat tolerance, the 0.25 ml aliquots of cell suspensionwere combined with 0.25 ml of SCD-Ura medium and incubated in 96-wellplates (2 ml square wells) at 42° C. All selective cultures wereincubated for 72 hours: those measuring heat tolerance at 42° C. and allothers at 30° C., with the exception of the butanol selections whichwere cultured for 48 hours. All selective cultures were incubated withconstant shaking at 800 rpm.

The cultures were then diluted 1:100 and 1:500 in SCD-Ura. 150 μlaliquots of the diluted cells were plated onto 10 cm plates containingsolid minimal uracil dropout medium containing glucose as a carbonsource (SCD-Ura agar; for 1 L medium, 6.7 g yeast nitrogen base, 0.77 guracil dropout mix and 15 g Bacto Agar are combined, the pH is adjustedto 5.6-5.8 with 120 μl 10N NaOH, and after autoclaving 20 g of glucoseare added as a sterile 20% solution). The plates were allowed to dry andwere incubated at 30° C. for 2 days to allow surviving cells to growinto colonies. Colonies were counted on each plate, and the averagenumber of surviving cells and their standard deviations (Std Dev) fromeach set of transformants were computed. Results are shown in Table 6(FIGS. 3 and 4) under the columns labeled ‘Number of surviving cells’.The number of starting cells in each culture was approximately 2.5×10⁶.

Yeast strain BY4741 transformed with various KEOPS/EGO TEF1 fusionpolynucleotide centromeric plasmids showed enhanced resistance to ortolerance of heat, ethanol, butanol and low pH, compared to theKEOPS/EGO TEF1 individual ORF centromeric plasmids, as shown in Table 6(FIGS. 3 and 4). Yeast strain BY4741 containing the negative controlplasmid p466-TEF1 (SEQ ID NO 128) served as a control.

Only one of the individual full length ORFs (WHI2) showed any stresstolerance activity and all the others had no measurable activity.Activities of the fusion polynucleotides that surpass the WHI2 score areidentified by grey shaded cells in Table 6 (FIGS. 3 and 4). In manycases, the activity level is significantly above that of the individualfull length WHI2 ORF, suggesting unexpected gain-of-function activity bythe fusion polynucleotide. Furthermore, the table shows that eachcomponent of the KEOPS complex, and each component of the EGO complex,led to enhanced activity of WHI2 in at least one fusion polynucleotideand phenotypic measurement, suggesting that all members of one complexare able to interact positively with the activity of WHI2 in a fusionprotein and/or cause unexpected gain-of-function tolerance or resistanceactivity when fused with WHI2.

All resistance and tolerance scores are listed in Table 6 which is splitinto two parts; part A (FIG. 3) shows cell counts from heat and ethanolselections and part B (FIG. 4) shows cell counts from the butanol andlow pH selections.

Example 7 Comparison of Resistance and Tolerance Phenotypes Conferred byFusion Polynucleotides and Individual ORFs in Galactose-ContainingMedium

Fourteen selected fusion polynucleotide GAL1 centromeric plasmids fromExample 2 were used to generate quantitative measurements for heat,ethanol, butanol and low pH resistance and/or tolerance activity. The 14fusion polynucleotides were compared in their activity to the 14individual full length ORFs that are the building blocks of the fusionpolynucleotides.

The 14 fusion polynucleotides and the 14 individual ORFs were clonedinto a derivative of the yeast centromeric plasmid p416-GAL1 (Mumberg1995, Funk 2002). The yeast centromeric plasmid p416-GAL1 contains thefollowing sequences for plasmid propagation in yeast and E. coli, andexpression of an inserted polynucleotide: the bacterial replicon ofplasmid pMB1; the bacterial ampicillin-resistance gene; the yeastCEN6/ARSH4 cassette (Sikorski 1989) containing the chromosome 6centromere and the yeast histone H4-associated autonomously replicatingsequence (ARS); the yeast URA3 prototrophic marker gene; and the yeastGAL1 promoter placed adjacent to the CYC1 terminator in a manner thatallows expression of coding regions placed between them (see SEQ ID NO:127). All polynucleotides were cloned between nucleotides 3206 and 3207of SEQ ID NO 127.

The resulting set of centromeric plasmids containing expressioncassettes of the 14 fusion polynucleotides are hereafter referred to asthe ‘fusion polynucleotide GAL1 centromeric plasmids;’ the centromericplasmids containing expression cassettes of the 14 individual ORFs arehereafter referred to as the ‘individual ORF GAL1 centromeric plasmids’.Collectively, the expression constructs are referred to as the ‘GAL1centromeric plasmids’.

All experiments measuring tolerance and resistance activities of theGAL1 centromeric plasmids were performed using strain BY4741 (Brachmann1998) transformed with each GAL1 centromeric plasmid. Yeasttransformations were performed using the lithium acetate-heat shockmethod as described in Example 2. The GAL1 centromeric plasmidtransformants of strain BY4741 were maintained on minimal uracil dropoutsolid medium containing glucose as a carbon source (SCD-Ura agar; for 1L medium, 6.7 g yeast nitrogen base, 0.77 g uracil dropout mix, and 15 gBacto Agar are combined, the pH is adjusted to 5.6-5.8 with 120 μl 10NNaOH and after autoclaving 20 g of glucose are added as a sterile 20%solution).

Resistance and/or tolerance phenotypes for heat (42° C.), ethanol (15%),butanol (3%), low pH (pH 3.0 in 0.2M sodium acetate) and salt (2M NaCl)were measured in triplicate for the 12 yeast strains shown in Table 7A(FIG. 5) with three independent transformants of yeast strain BY4741with each GAL1 centromeric plasmid. The 13 strains were first culturedin 96-well plates (2 ml square wells) in synthetic complete uracildropout medium containing glucose as a carbon source (SCD-Ura; for 1 Lmedium, 6.7 g yeast nitrogen base and 0.77 g uracil dropout mix arecombined, the pH is adjusted to 5.6-5.8 with 120 μl 10N NaOH, and afterautoclaving 20 g of glucose are added as a sterile 20% solution). Cellswere incubated for 16 hours at 30° C. with constant shaking at 800 rpmwith a 3 mm radius of gyration.

The cell densities were measured with a hemocytometer and the cellsuspensions diluted to a final cell density of 1×10⁷ cells/ml in 1.25 mlrich medium containing galactose as a carbon source (YPGal; for 1 Lmedium, 20 g Bacto Peptone and 10 g Bacto Yeast Extract are combined andafter autoclaving 20 g of galactose are added as a sterile 20%solution). The diluted cultures were incubated for an additional 5 hoursunder the same conditions. The cell suspensions were then added in 0.25ml aliquots to fresh 96-well plates (2 ml square wells) and combinedwith 0.25 ml per well of fresh YPGal containing a 2× concentration ofone selective agent. The selective agents used were: NaCl at a finalconcentration of 2M (salt selection); ethanol at a final concentrationof 15%; n-butanol at a final concentration of 3%; or sodium acetate pH3.0 at a final concentration of 0.2M. The sodium acetate was prepared asa 1M solution of sodium acetate pH 3.0 by mixing 2.74 ml glacial aceticacid with 0.11 g anhydrous sodium acetate in 50 ml final volume andfilter sterilized using a 0.2 micron filter. For measuring heattolerance, the 0.25 ml aliquots of cell suspension were combined with0.25 ml of YPGal and cultured in 96-well plates (2 ml square wells) at42° C. All selective cultures were incubated for 3 days with constantshaking at 800 rpm; those measuring heat tolerance at 42° C. and allothers at 30° C.

The cultures for Table 7A (FIG. 5) were then spotted undiluted or in1:10 dilutions (diluted in SCD-Ura) onto fresh 15 cm plates containingsolid rich medium with glucose as a carbon source (YPD agar; for 1 Lmedium, 20 g Bacto Peptone, 10 g Bacto Yeast Extract and 15 g Bacto Agarare combined, and after autoclaving 20 g glucose are added as a 20%sterile solution). The spotting was done using a Bel-Art 96-wellreplicating tool (Bel-Art Products) that deposits spots of approximately3 μl from each well onto the recipient plate. The spots were allowed todry, the plates incubated at 30° C. for 1 day to allow surviving cellsto grow into cell spots or individual colonies, and the cell density ofeach transformant for each selective condition scored on a relativescale from 0-3; 0 being no growth, 1 slight growth, 2 significant growthand 3 confluent growth. Both the undiluted and diluted spots were takeninto account to generate the score. To obtain a final activity score foreach fusion polynucleotide GAL1 centromeric plasmid and each selectivecondition, the triplicate scores for each selective condition wereadded, and the added triplicate score obtained with the control plasmidsubtracted, and the result divided by the number of replicates (3).Final activity scores below zero were scored as zero. All scores areshown in Table 7A (FIG. 5) under the columns labeled ‘Activity scores’.

Resistance and tolerance phenotypes of the 20 yeast strains shown inTable 7B (FIG. 6) to heat (42° C.), ethanol (15%), butanol (3%), low pH(pH 3.0 in 0.2M sodium acetate) and salt (2M NaCl) were measured inquadruplicate with four independent transformants of yeast strain BY4741with each GAL1 centromeric plasmid. The strains were first grown in96-well plates (2 ml square wells) containing minimal uracil dropoutmedium containing 2% raffinose as a carbon source (SCRaf-Ura; for 1 Lmedium, 6.7 g yeast nitrogen base and 0.77 g uracil dropout mix arecombined, pH is adjusted to 5.6-5.8 with 120 μl 10N NaOH, and afterautoclaving 20 g of raffinose are added as a sterile 20% solution).Cells were incubated for 16 hours at 30° C. with constant shaking at 800rpm on a microshaker with a 3 mm radius of gyration.

The cell densities were then measured with a hemocytometer, and the cellsuspensions diluted to a final cell density of 1×10⁷ cells/ml in 1.25rich medium containing raffinose and galactose as carbon sources(YPRaf-Gal; for1 L medium,20 g Bacto Peptone and 10 g Bacto YeastExtract are combined and after autoclaving 20 g of raffinose and 20 g ofgalactose are added as sterile 20% solutions). The cell suspensions tofresh 96-well plates (2 ml square wells) and cultured for an additional2 hours in the same manner. The cultures were then were then added in0.25 ml aliquots to fresh 96-well plates (2 ml square wells) containing0.25 ml per well of YPRaf-Gal with 2× concentration of one selectiveagent. The selective agents used were 15% final concentration ofethanol, 3% final concentration of n-butanol, 0.2M final concentrationof sodium acetate pH 3.0 or 2M final concentration of NaCl. The sodiumacetate was prepared as a 1M solution of sodium acetate pH 3.0 by mixing2.74 ml glacial acetic acid with 0.11 g anhydrous sodium acetate in 50ml final volume and filter sterilized using a 0.2 micron filter Formeasuring heat tolerance, the 0.25 ml aliquots of cell suspension werecombined with 0.25 ml of YPRaf-Gal and cultured in 96-well plates (2 mlsquare wells) at 42° C. All selective cultures were incubated for 48hours with constant shaking at 800 rpm; those measuring heat toleranceat 42° C. and all others at 30° C.

The cultures for Table 7B (FIG. 6) were then spotted undiluted or in1:10 dilutions (diluted in SCD-Ura) onto fresh 15 cm plates containingsolid rich medium with glucose as a carbon source (YPD agar; for 1 Lmedium, 20 g Bacto Peptone, 10 g Bacto Yeast Extract and 15 g Bacto Agarare combined, and after autoclaving 20 g glucose are added as a sterile20% solution). The cultures were spotted using a Bel-Art ProductsBel-Art 96-well replicating tool that deposits spots of approximately 3μl from each well onto the recipient plate. The spots were allowed todry, the plates incubated at 30° C. for 1 day to allow surviving cellsto grow into cell spots or individual colonies, and the cell density ofeach transformant for each selective condition scored on a relativescale from 0-3; 0 being no growth, 1 slight growth, 2 significant growthand 3 confluent growth. Both the undiluted and diluted spots were takeninto account to generate the score. To obtain a final activity score foreach fusion polynucleotide GAL1 centromeric plasmid for each selectivecondition, the quadruplicate scores for each selective condition wereadded, and the added score obtained with the control plasmid subtracted,and the result divided by the number of replicates (4). Final activityscores below zero were scored as zero. All scores are shown in Table 7B(FIG. 6) under the columns labeled ‘Activity scores’.

Yeast strain BY4741 transformed with the fusion polynucleotide GAL1centromeric plasmids showed enhanced resistance to or tolerance of heat,ethanol, butanol, low pH and/or salt, compared to the individual ORFGAL1 centromeric plasmids, as shown in Table 7 (FIGS. 5 and 6). Yeaststrain BY4741 containing the negative control plasmid p416-GAL1 (Mumberg1995, Funk 2002) served as a control.

From the stress tolerance activities of the individual ORFs composingeach fusion polynucleotide, the maximal expected stress tolerance scorefor each fusion polynucleotide can easily be calculated by adding theindividual stress tolerance scores of each ORF contained in the fusionpolynucleotide. Activities of the fusion polynucleotides that surpassthis additive score are identified by grey shaded cells in Table 7(FIGS. 5 and 6). The activity of the fusion polynucleotide would beexpected to be equivalent to the individual value of one of thecomponent ORFs and perhaps, in exceptional cases, to approach theadditive score of the two component ORFs. However, the table shows thatmany of the fusion polynucleotide activities are even higher than theadditive values. Fusion polynucleotide activity surpassed the additiveactivity levels in 19 out of 30 cases in Table 7A (FIG. 5) and 24 out of45 cases in Table 7B (FIG. 6).

In one or more of the phenotypic categories (heat, ethanol, butanol, lowpH and salt tolerance), each fusion polynucleotide listed in Table 7(FIGS. 5 and 6) shows unexpected gain-of-function activity that iscreated by fusing together the two component ORFs. However, theexpectation would have been that the activity of each fusionpolynucleotide approximates the activity of one of the starting ORFs, orat best matches the additive activities of the two ORFs.

Activity scores of 14 fusion polynucleotide GAL1 centromeric plasmidsand their 14 individual component ORFs are shown in Table 7 (FIGS. 5 and6).

Example 8 Resistance and Tolerance Phenotypes in Glucose-ContainingMedia of Yeast Strains Transformed with Fusion PolynucleotideCentromeric Plasmids

All 83 fusion polynucleotides (SEQ ID NOs 1-63 and 205-224) are clonedinto the yeast expression vector p466-TEF1 (SEQ. ID No 128) which issimilar to the yeast centromeric plasmid p416-TEF1 (Mumberg 1995, Funk2002). The yeast centromeric plasmid p466-TEF1 contains the followingsequences for plasmid propagation in yeast and E. coli, and expressionof an inserted polynucleotide: the bacterial replicon of plasmid pMB1;the bacterial ampicillin-resistance gene; the yeast CEN6/ARSH4 cassette(Sikorski 1989) containing the chromosome 6 centromere and the yeasthistone H4-associated autonomously replicating sequence (ARS); the yeastURA3 prototrophic marker gene; and the yeast TEF1 promoter placedadjacent to the CYC1 terminator in a manner that allows expression ofcoding regions inserted between them. The sequence of this p466-TEF1expression vector is given in SEQ ID NO 128. All polynucleotides arecloned between nucleotide numbers 3029 and 3030 of SEQ ID NO 128.

The resulting set of constructs, referred to as ‘fusion polynucleotideTEF1 centromeric plasmids’, contain expression cassettes of the 83fusion polynucleotides that are given in SEQ ID NOs: 1-63, and 205-224under control of the S. cerevisiae TEF1 promoter. These constructs areused for quantitative determinations of yeast tolerance to the followingstresses and toxic agents: alcohols (propanol, isopropanol, iso-butanol,pentanol, hexanol, heptanol, octanol, nonanol, decanol, undecanol anddodecanol), alkanes (butane, pentane, hexane, heptane, octane, nonane,decane, undecane, dodecane, hexadecane), heavy metal ions (various ionsof the elements Ag, silver; As, arsenic; Cd, cadmium; Cr, chromium; Co,cobalt; Cu, copper; Hg, mercury; Ni, nickel; Pb, lead; Pt, platinum; Sb,antimony; Se, selenium; Tl, thallium; or Zn, zinc), oxidizing agents(hydrogen peroxide and other inorganic peroxides; inorganic oxidizingagents such as potassium permanganate; organic peroxides andhydroperoxides such as ethyl hydroperoxide, diacetyl peroxide, diethylmaleate, tert-butyl hydroperoxide, cumyl hydroperoxide and ascaridole;and other toxic compounds such as furfural, 5-hydroxymethylfurfural,benzoic acid derivatives (for example p-hydroxybenzoic acid), and othertoxic lignin breakdown products found in sugar preparations derived frombiomass (Luo 2002).

All experiments measuring tolerance and resistance activities of thefusion polynucleotides are performed using strain BY4741 (Brachmann1998) transformed with each fusion polynucleotide TEF1 centromericplasmid. Yeast transformations are performed using the lithiumacetate-heat shock method as described in Example 2. The individualfusion polynucleotide TEF1 centromeric plasmid transformants of strainBY4741 are maintained on minimal uracil dropout solid medium containingglucose as a carbon source (SCD-Ura agar; for 1 L medium, 6.7 g yeastnitrogen base, 0.77 g uracil dropout mix, and 15 g Bacto Agar arecombined, the pH is adjusted to 5.6-5.8 with 120 μl 10N NaOH and afterautoclaving 20 g of glucose are added as a sterile 20% solution).

The tolerance determinations are performed either in rich mediumcontaining glucose as a carbon source or in minimal medium containingglucose as a carbon source. The tolerance activities of each fusionpolynucleotide TEF1 centromeric plasmid are measured in quadruplicate,with each of two independent transformants of each fusion polynucleotideTEF1 centromeric plasmid tested in duplicate.

Selective concentrations for all selective agents are determined bydiluting an overnight culture of yeast strain BY4741 (Brachmann 1998) toa density of 5×10⁶ cells/ml in rich medium containing glucose as acarbon source (YPD; for 1 L medium, 20 g Bacto Peptone and 10 g BactoYeast Extract are combined and after autoclaving 20 g glucose are addedas a 20% sterile solution) or minimal medium containing glucose as acarbon source (SCD-Ura; for 1 L medium, 6.7 g yeast nitrogen base, 0.77g uracil dropout mix and 25 ml of 2 mg/ml uracil are combined, the pH isadjusted to 5.6-5.8 with 120 μl 10N NaOH and after autoclaving 20 g ofglucose are added as a sterile 20% solution). The cells are pre-culturedby adding 0.25 ml of the cell suspension to each well of a 96-well plate(2 ml square wells) and incubating for 2 hours at 30° C. with constantshaking at 800 rpm with a 3 mm radius of gyration. To each well, 0.25 mlof the same fresh medium is then added containing differentconcentrations of the selective agents. Selective concentrations foralcohols range between 0.025% and 25% depending on alcohol chain length(Fujita 2004). Selective concentrations for alkanes range between 1% and25% depending on alkane chain length (Gill 1972, Fujita 2004); selectiveconcentrations for heavy metal ions range between 0.5 μM and 10 mM(Bitton 1984, Hsu 1992, Nguyên-nhu 2002, Soares 2003); selectiveconcentrations for oxidizing agents range from 2-100 mM for hydrogenperoxide, other inorganic peroxides and potassium permanganate, and0.5-10 mM for organic peroxides and hydroperoxides (Krems 1995,Nguyên-nhu 2002, Miyazaki 2005); selective concentrations for furfural,hydroxymethylfurfural and p-hydroxybenzoic acid range from 2-100 mM(Palmqvist 1999, Liu 2005, Liu 2006, Heer 2008, Liu 2008, Tofighi 2010).The selective plates are sealed and incubated at 30° C. shaking at 800rpm with a 3 mm radius of gyration for 48-96 hours. The cell density ofeach culture is then measured with a hemocytometer, and the growthinhibitory concentration of each compound determined. Cells from eachgrowth inhibited culture are then plated on fresh YPD solid medium (YPDagar; for 1 L medium, 20 g Bacto Peptone, 10 g Bacto Yeast Extract and15 g Bacto Agar are combined and after autoclaving 20 g glucose areadded as a 20% sterile solution) and allowed to grow for 1-2 days toassess the relative number of viable cells in each culture and todetermine the lethal concentration of each compound. The compounds arethen divided into lethal and growth-inhibitory, with separate toleranceand resistance assays conducted for each class.

For measurement of the resistance to and tolerance of lethal compoundsamong the alcohols, alkanes, heavy metal ions, oxidizing agents andother toxic compounds listed above, the 84 yeast strains (BY4741transformed with 83 fusion polynucleotide TEF1 centromeric plasmids plusone control vector) are first cultured in 96-well plates (2 ml squarewells) in 1 ml per well of SCD-Ura. Cells are incubated for 16 hours at30° C. with constant shaking at 800 rpm with a 3 mm radius of gyration.The cell densities are then measured with a hemocytometer and the cellsdiluted in a fresh 96-well plate (2 ml square wells) to a final celldensity of 1×10⁷ cells/ml in 1.25 ml SCD-Ura or YPD medium, and grownfor an additional 2 hours. The cell suspensions are then added in 0.25ml aliquots to fresh 96-well plates (2 ml square wells) and combinedwith 0.25 ml per well of fresh YPD medium or SCD-Ura medium, the freshlyadded medium containing 2× concentration of one selective agent. Theselective agents used are listed above, and lethal concentrations ofeach are used in the selection. All selective cultures are cultured for48-96 hours at 30° C. After the selection has been completed, thecultures are then diluted 1:10, 1:100 and 1:1000 in SCD-Ura liquidmedium and 150 μl aliquots of the diluted cells are plated as ˜1 inspots onto 15 cm plates containing SCD-Ura agar medium or YPD agarmedium. The plates are allowed to dry and are incubated at 30° C. for 2days to allow surviving cells to grow into colonies. The colonies arecounted, the multiple counts for each fusion polynucleotide TEF1centromeric plasmid are averaged and standard deviations are calculated,to determine the relative stress tolerance and resistance phenotypesconferred by each fusion polynucleotide.

For measurement of the resistance to and tolerance of growth-inhibitorycompounds among the alcohols, alkanes, heavy metal ions, oxidizingagents and other toxic compounds listed above, the 84 yeast strains(BY4741 transformed with 83 fusion polynucleotide TEF1 centromericplasmid plus one control vector) are first cultured in 96-well plates (2ml square wells) in 1 ml per well of SCD-Ura. Cells are incubated for 16hours at 30° C. with constant shaking at 800 rpm with a 3 mm radius ofgyration. The cell density is then measured with a hemocytometer, andthe cells diluted in a fresh 96-well plate (2 ml square wells) to afinal cell density of 1×10⁷ cells/ml in 1.25 ml SCD-Ura or YPD medium,and grown for an additional 2 hours. The cell suspensions are then addedin 0.25 ml aliquots to fresh 96-well plates (2 ml square wells) andcombined with 0.25 ml per well of fresh SCD-Ura or YPD medium, thefreshly added medium containing 2× concentration of one selective agent.The selective agents used are listed above, and growth-inhibitoryconcentrations of each are used in the selection. All selective culturesare cultured for 48-96 hours at 30° C. After the selection has beencompleted, the cultures are diluted 1:100, 1:1000 and 1:10,000 inSCD-Ura liquid medium and 150 μl aliquots of the diluted cells areplated as ˜1 in spots onto 15 cm plates containing SCD-Ura agar mediumor YPD agar medium. The plates are allowed to dry and are incubated at30° C. for 2 days to allow surviving cells to grow into colonies. Thecolonies are counted, the multiple counts for each fusion polynucleotideTEF1 centromeric plasmid are averaged and used to calculate the numberof viable cells in each culture as well as standard deviations, todetermine the relative stress tolerance and resistance phenotypesconferred by each fusion polynucleotide.

Example 9 Efficient Method for Selection of Heat-Tolerant Yeast Clones

A collection or library of Saccharomyces cerevisiae in-frame fusionpolynucleotides is prepared as described in U.S. patent application Ser.No. 14/134,619 and International Patent Application Serial NumberPCT/US13/76526. The randomized in-frame fusion polynucleotides arecloned into a vector molecule, such the yeast expression vectorp416-GAL1 (Funk 2002). The sequence of a p416-GAL1 derivative vector isgiven in SEQ ID NO 127 and contains the following sequences for plasmidpropagation in yeast and E. coli and expression of an insertedpolynucleotide: the bacterial replicon of plasmid pMB1, the bacterialampicillin/carbenicillin-resistance gene, the yeast CEN6/ARSH4 cassette(Sikorski 1989) containing the chromosome 6 centromere and the yeasthistone H4-associated autonomously replicating sequence (ARS), the yeastURA3 prototrophic marker gene, and the yeast GAL1 promoter and CYC1terminator placed adjacent to each other in a manner that allowsinsertion of coding regions in between to allow their expression. Allrandomized in-frame fusion polynucleotides are cloned between nucleotidenumbers 3206 and 3207 of SEQ ID NO 127.

Yeast transformations are performed by the lithium acetate-heat shockmethod (Gietz 2002, Gietz 2006, Gietz 2007). The yeast strain BY4741(Brachmann 1998) from a plate or an overnight culture is inoculated into50 ml of YPD medium (for 1 L medium, 20 g Bacto Peptone, 10 g BactoYeast Extract are combined and after autoclaving 20 g Glucose are addedas a 20% sterile solution) at 30° C. on a shaker at 225 rpm from astarting density of 5×10⁶ cells/ml (cell density determined with ahemocytometer), and grown over several hours to a final cell density of2×10⁷ cells/ml. The cells are harvested by centrifuging at 3000 g for 5min, are then resuspended in 25 ml of sterile deionized water, andcentrifuged again. Cells are resuspended in 1 ml of sterile water,transferred to a 1.5 ml microcentrifuge tube, centrifuged for 30 sec at3000 rpm and the supernatant aspirated. The cell pellet is thenresuspended in 0.4 ml of sterile deionized water. The cell suspension iscombined with 3.26 ml of transformation mix (2.4 ml of 50% w/v PEG 3350,360 μl 1M Lithium acetate and 500 μl 10 mg/ml sheared, boiled salmonsperm DNA) and mixed well. Aliquots of DNA (100 ng-1 μg) are pipettedinto separate 1.5 ml microcentrifuge tubes and combined with 380 μl ofthe cell suspension in transformation mix. The cell/DNA mixture is mixedthoroughly and is incubated at 42° C. on a shaker at 250 rpm for 40minutes. The transformations are then centrifuged for 1 minute at 3000rpm in a microcentrifuge, the supernatant aspirated and each cellaliquot resuspended in 0.5-1 ml sterile deionized water. Depending onthe desired density of colonies, 10 μl to 1 ml of the cell suspensionare plated with sterile 4 mm glass beads onto one 10 cm or 15 cm platecontaining synthetic complete uracil dropout solid medium having glucoseas a carbon source (SCD-Ura agar; for 1 L, 6.7 g yeast nitrogen base,0.77 g uracil dropout mix and 15 g Bacto agar are combined, the pH isadjusted with 120 μl 10N NaOH to bring the pH to 5.6-5.8, and afterautoclaving 20 g glucose are added as a 20% sterile solution). Afterdrying, the plates are covered and incubated at 30° C. for 3-4 daysuntil colonies of transformants have formed.

After formation of colonies or lawns of cells transformed withrandomized in-frame fusion polynucleotides, the transformed cells areremoved from the selective plates by scraping with glass beads. This isdone by adding to each 10 cm plate 5 ml synthetic complete uracildropout medium with galactose as a carbon source (SCGal-Ura; for 1 Lmedium, 6.7 g yeast nitrogen base and 0.77 g uracil dropout mix arecombined, the pH is adjusted with 120 μl 10N NaOH to bring the pH to5.6-5.8 and, after autoclaving, 20 g galactose are added as a sterile20% solution) together with 10×4 mm glass beads. Proportionally highervolumes of medium are added to larger plates. Using swirling andhorizontal shaking motions to allow the glass beads to dislodge theyeast cells from the surface of the agar, the resuspended cells arecollected with a pipet, using additional medium if desired to wash anyremaining cells off the plate. Cells collected in this fashion arepelleted by centrifugation at 4000 rpm for 5 minutes. Cells areresuspended in SCGal-Ura at a cell density of 5×10⁶ cells/ml andcultured at 30° C. shaking at 250 rpm for 4-12 hours. This pre-culturingstep allows induction of the GAL1 promoter used to express therandomized in-frame fusion polynucleotides.

For heat tolerance selection, tolerant cells are selected in liquidculture. The cell density in the suspension of yeast transformantscontaining in-frame fusion polynucleotides is determined with ahemocytometer, and the cells are suspended in SCGal-Ura at a celldensity of 5×10⁶ cells/ml in 50 ml of medium in a 500 ml flask andcultured at 40-44° C. shaking at 250 rpm for 7 days. This selectiveculture is referred to as the ‘heat liquid selection culture’. Followingthe selection, 0.5 ml aliquots of the cell suspension are either plateddirectly on 15 cm SCD-Ura agar plates to select for surviving cells, orthe cell suspension after selection is centrifuged at 3000 rpm for 5minutes, the cell pellet suspended in 0.25 ml minimal uracil dropoutsolid medium with glucose as a carbon source (SCD-Ura; for 1 L, 6.7 gyeast nitrogen base and 0.77 g uracil dropout mix are combined, the pHis adjusted with 120 μl 10N NaOH to bring the pH to 5.6-5.8, and afterautoclaving 20 g glucose are added as a 20% sterile solution) and platedon a 15 cm SCD-Ura agar plate. The plates are allowed to dry andincubated at 30° C. for 3-4 days until colonies have formed.

To boost the number of survivors from heat selection, it is advantageousto incubate the heat liquid selection culture at temperatures rangingfrom 4° C. to 25° C. either with or without shaking for 1-24 hours afterremoval from the heat selection. For example, following a heat selectionat 42° C., the heat liquid selection culture is removed from the shakerin which the selection took place and transferred to room temperaturewhere it is allowed to remain without shaking for 2 hours. The heatliquid selection culture is then incubated at 4° C. for 16 hours. Thecells are then collected centrifuged at 3000 rpm for 5 minutes, the cellpellet suspended in 0.25 ml SCD-Ura and plated on a 15 cm SCD-Ura agarplate. The plate is allowed to dry and incubated at 30° C. for 3-4 daysuntil colonies have formed. Comparison between the number of coloniesformed when plating directly after heat selection, vs plating aftersuccessive incubation of the heat liquid selection culture at roomtemperature and 4° C. shows a greater number of colonies aftersuccessive incubation at room temperature and 4° C. by a factor of 2-50over the control.

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All publications, databases, GenBank sequences, patents and patentapplications cited in this Specification are herein incorporated byreference as if each was specifically and individually indicated to beincorporated by reference.

We claim:
 1. A polypeptide comprising an amino acid sequence having atleast 90% sequence identity to SEQ ID NO:
 110. 2. The polypeptideaccording to claim 1, wherein the sequence identity is at least 95%. 3.The polypeptide according to claim 1, wherein the sequence comprises SEQID NO.
 110. 4. The polypeptide according to claim 1, wherein thesequence consists of SEQ ID NO:110.